The Role of Neurod Genes in Brain Development, Function, and Disease

Transcriptional regulation is essential for the correct functioning of cells during development and in postnatal life. The basic Helix-loop-Helix (bHLH) superfamily of transcription factors is well conserved throughout evolution and plays critical roles in tissue development and tissue maintenance. A subgroup of this family, called neural lineage bHLH factors, is critical in the development and function of the central nervous system. In this review, we will focus on the function of one subgroup of neural lineage bHLH factors, the Neurod family. The Neurod family has four members: Neurod1, Neurod2, Neurod4, and Neurod6. Available evidence shows that these four factors are key during the development of the cerebral cortex but also in other regions of the central nervous system, such as the cerebellum, the brainstem, and the spinal cord. We will also discuss recent reports that link the dysfunction of these transcription factors to neurological disorders in humans.

The initiation of Otx2 expression in the developing mouse retina requires a unique enhancer and either Ascl1 or Neurog2 activity

During retinal development, a large subset of progenitors upregulates the transcription factor Otx2, which is required for photoreceptor and bipolar cell formation. How these retinal progenitor cells initially activate Otx2 expression is unclear. To address this, we investigated the cis-regulatory network that controls Otx2 expression. We identified a minimal enhancer element, DHS-4D, that drove expression in newly formed OTX2+ cells. CRISPR/Cas9 mediated deletion of DHS-4D reduced OTX2 expression, but this effect was diminished in postnatal development. Systematic mutagenesis of the enhancer revealed that three basic helix-loop-helix (bHLH) transcription factor binding sites were required for its activity. Single cell RNA-sequencing of nascent Otx2+ cells identified the bHLH factors Ascl1 and Neurog2 as candidate regulators. CRISPR/Cas9 targeting of these factors showed that only the simultaneous loss of Ascl1 and Neurog2 prevented OTX2 expression. Our findings suggest that Ascl1 and Neurog2 act redundantly or in a compensatory fashion to activate the DHS-4D enhancer and Otx2 expression. We observed redundancy or compensation at both the transcriptional and enhancer utilization levels, suggesting that the mechanisms governing Otx2 regulation in the retina are flexible and robust.

Single Cell Transcriptomic Analyses Reveal the Impact of bHLH Factors on Human Retinal Organoid Development

The developing retina expresses multiple bHLH transcription factors. Their precise functions and interactions in uncommitted retinal progenitors remain to be fully elucidated. Here, we investigate the roles of bHLH factors ATOH7 and Neurog2 in human ES cell-derived retinal organoids. Single cell transcriptome analyses identify three states of proliferating retinal progenitors: pre-neurogenic, neurogenic, and cell cycle-exiting progenitors. Each shows different expression profile of bHLH factors. The cell cycle-exiting progenitors feed into a postmitotic heterozygous neuroblast pool that gives rise to early born neuronal lineages. Elevating ATOH7 or Neurog2 expression accelerates the transition from the pre-neurogenic to the neurogenic state, and expands the exiting progenitor and neuroblast populations. In addition, ATOH7 and Neurog2 significantly, yet differentially, enhance retinal ganglion cell and cone photoreceptor production. Moreover, single cell transcriptome analyses reveal that ATOH7 and Neurog2 each assert positive autoregulation, and both suppress key bHLH factors associated with the pre-neurogenic and states and elevate bHLH factors expressed by exiting progenitors and differentiating neuroblasts. This study thus provides novel insight regarding how ATOH7 and Neurog2 impact human retinal progenitor behaviors and neuroblast fate choices.

SPEECHLESS and MUTE Mediate Feedback Regulation of Signal Transduction during Stomatal Development

Stomatal density, spacing, and patterning greatly influence the efficiency of gas exchange, photosynthesis, and water economy. They are regulated by a complex of extracellular and intracellular factors through the signaling pathways. After binding the extracellular epidermal patterning factor 1 (EPF1) and 2 (EPF2) as ligands, the receptor-ligand complexes activate by phosphorylation through the MAP-kinase cascades, regulating basic helix-loop-helix (bHLH) transcription factors SPEECHLESS (SPCH), MUTE, and FAMA. In this review, we summarize the molecular mechanisms and signal transduction pathways running within the transition of the protodermal cell into a pair of guard cells with a space (aperture) between them, called a stoma, comprising asymmetric and symmetric cell divisions and draw several functional models. The feedback mechanisms involving the bHLH factors SPCH and MUTE are not fully recognized yet. We show the feedback mechanisms driven by SPCH and MUTE in the regulation of EPF2 and the ERECTA family. Intersections of the molecular mechanisms for fate determination of stomatal lineage cells with the role of core cell cycle-related genes and stabilization of SPCH and MUTE are also reported.

Maternal High Fat Diet Programs Male Mice Offspring Hyperphagia and Obesity: Mechanism of Increased Appetite Neurons via Altered Neurogenic Factors and Nutrient Sensor AMPK

Maternal high-fat (HF) is associated with offspring hyperphagia and obesity. We hypothesized that maternal HF alters fetal neuroprogenitor cell (NPC) and hypothalamic arcuate nucleus (ARC) development with preferential differentiation of neurons towards orexigenic (NPY/AgRP) versus anorexigenic (POMC) neurons, leading to offspring hyperphagia and obesity. Furthermore, these changes may involve hypothalamic bHLH neuroregulatory factors (Hes1, Mash1, Ngn3) and energy sensor AMPK. Female mice were fed either a control or a high fat (HF) diet prior to mating, and during pregnancy and lactation. HF male newborns were heavier at birth and exhibited decreased protein expression of hypothalamic bHLH factors, pAMPK/AMPK and POMC with increased AgRP. As adults, these changes persisted though with increased ARC pAMPK/AMPK. Importantly, the total NPY neurons were increased, which was consistent with the increased food intake and adult fat mass. Further, NPCs from HF newborn hypothalamic tissue showed similar changes with preferential NPC neuronal differentiation towards NPY. Lastly, the role of AMPK was further confirmed with in vitro treatment of Control NPCs with pharmacologic AMPK modulators. Thus, the altered ARC development of HF offspring results in excess appetite and reduced satiety leading to obesity. The underlying mechanism may involve AMPK/bHLH pathways.

Single cell transcriptomic analyses reveal the impact of bHLH factors ATOH7 and Neurog2 on human retinal organoid development

The developing retina expresses multiple bHLH transcription factors. Their precise functions and interactions in uncommitted retinal progenitors remain to be fully elucidated. Here, we investigate the roles of bHLH factors ATOH7 and Neurog2 in developing human ES cell-derived 3D retinal organoids. Single cell transcriptome analyses identify three states of proliferating retinal progenitors: pre-neurogenic, neurogenic, and cell cycle-exiting progenitors. Each shows different expression profile of bHLH factors. The distinct cell cycle-exiting progenitors feed into a postmitotic heterozygous neuroblast pool that gives rise to early born neuronal lineages. Elevating ATOH7 or Neurog2 expression accelerates the transition from the pre-neurogenic to the neurogenic state, and expands the exiting progenitor and neuroblast populations. In addition, ATOH7 and Neurog2 significantly, yet differentially, enhance retinal ganglion cell and cone photoreceptor production. Moreover, single cell transcriptome analyses reveal that ATOH7 and Neurog2 assert positive autoregulation, suppress key bHLH factors associated with the neurogenic progenitors, and elevate bHLH factors expressed by exiting progenitors and differentiating neuroblasts. This study thus provides novel insight regarding how ATOH7 and Neurog2 impact human retinal progenitor behaviors and neuroblast fate choices.