bHLH Factor Research Articles

Viral-mediated Oct4 overexpression and inhibition of Notch signaling synergistically induce neurogenic competence in mammalian Müller glia.

Retinal Müller glia in cold-blooded vertebrates can reprogram into neurogenic progenitors to replace neurons lost to injury, but mammals lack this ability. While recent studies have shown that transgenic overexpression of neurogenic bHLH factors and glial-specific disruption of NFI family transcription factors and Notch signaling induce neurogenic competence in mammalian Müller glia, induction of neurogenesis in wildtype glia has thus far proven elusive. Here we report that viral-mediated overexpression of the pluripotency factor Oct4 ( Pou5f1 ) induces transdifferentiation of wildtype mouse Müller glia into bipolar neurons and stimulates this process synergistically in parallel with Notch loss of function. Single cell multiomic analysis shows that Oct4 overexpression leads to widespread changes in gene expression and chromatin accessibility, inducing activity of both the neurogenic transcription factor Rfx4 and the Yamanaka factors Sox2 and Klf4. This study demonstrates that viral-mediated overexpression of Oct4 induces neurogenic competence in wildtype retinal Müller glia, identifying mechanisms that could be used in cell-based therapies for treating retinal dystrophies.

NEUROD1: transcriptional and epigenetic regulator of human and mouse neuronal and endocrine cell lineage programs.

Transcription factors belonging to the basic helix-loop-helix (bHLH) family are key regulators of cell fate specification and differentiation during development. Their dysregulation is implicated not only in developmental abnormalities but also in various adult diseases and cancers. Recently, the abilities of bHLH factors have been exploited in reprogramming strategies for cell replacement therapy. One such factor is NEUROD1, which has been associated with the reprogramming of the epigenetic landscape and potentially possessing pioneer factor abilities, initiating neuronal developmental programs, and enforcing pancreatic endocrine differentiation. The review aims to consolidate current knowledge on NEUROD1's multifaceted roles and mechanistic pathways in human and mousecell differentiation and reprogramming, exploring NEUROD1 roles in guiding the development and reprogramming of neuroendocrine cell lineages. The review focuses on NEUROD1's molecular mechanisms, its interactions with other transcription factors, its role as a pioneer factor in chromatin remodeling, and its potential in cell reprogramming. We also show a differential potential of NEUROD1 in differentiation of neurons and pancreatic endocrine cells, highlighting its therapeutic potential and the necessity for further research to fully understand and utilize its capabilities.

Robust reprogramming of glia into neurons by inhibition of Notch signaling and nuclear factor I (NFI) factors in adult mammalian retina.

Generation of neurons through direct reprogramming has emerged as a promising therapeutic approach for treating neurodegenerative diseases. In this study, we present an efficient method for reprogramming retinal glial cells into neurons. By suppressing Notch signaling by disrupting either Rbpj or Notch1/2, we induced mature Müller glial cells to reprogram into bipolar- and amacrine-like neurons. We demonstrate that Rbpj directly activates both Notch effector genes and genes specific to mature Müller glia while indirectly repressing expression of neurogenic basic helix-loop-helix (bHLH) factors. Combined loss of function of Rbpj and Nfia/b/x resulted in conversion of nearly all Müller glia to neurons. Last, inducing Müller glial proliferation by overexpression of dominant-active Yap promotes neurogenesis in both Rbpj- and Nfia/b/x/Rbpj-deficient Müller glia. These findings demonstrate that Notch signaling and NFI factors act in parallel to inhibit neurogenic competence in mammalian Müller glia and help clarify potential strategies for regenerative therapies aimed at treating retinal dystrophies.

FER-like iron deficiency-induced transcription factor (FIT) accumulates in nuclear condensates.

The functional importance of nuclear protein condensation remains often unclear. The bHLH FER-like iron deficiency-induced transcription factor (FIT) controls iron acquisition and growth in plants. Previously described C-terminal serine residues allow FIT to interact and form active transcription factor complexes with subgroup Ib bHLH factors such as bHLH039. FIT has lower nuclear mobility than mutant FITmSS271AA. Here, we show that FIT undergoes a light-inducible subnuclear partitioning into FIT nuclear bodies (NBs). Using quantitative and qualitative microscopy-based approaches, we characterized FIT NBs as condensates that were reversible and likely formed by liquid-liquid phase separation. FIT accumulated preferentially in NBs versus nucleoplasm when engaged in protein complexes with itself and with bHLH039. FITmSS271AA, instead, localized to NBs with different dynamics. FIT colocalized with splicing and light signaling NB markers. The NB-inducing light conditions were linked with active FIT and elevated FIT target gene expression in roots. FIT condensation may affect nuclear mobility and be relevant for integrating environmental and Fe nutrition signals.

Transplantation of Wnt3a-modified neural stem cells promotes neural regeneration and functional recovery after spinal cord injury via Wnt-Gli2 pathway.

Neural stem cell (NSCs) transplantation has great potential in the treatment of spinal cord injury (SCI). Previous studies have indicated that the Wnt pathway could regulate the expression of basic helix-loop-helix (bHLH) family factor Hes5 and Mash1 in NSCs, but not through the notch intracellular domain. This suggests that there are other signals involved in this process. The aim of this study was to investigate the role of Wnt-Gli2 pathway in the treatment of SCI by transplanting neural stem cells. NSCs were isolated from the striata of embryonic day 14 mice. Activation of the Wnt pathway was achieved using Wnt3a protein, while Gli2 was inhibited using Gli2-siRNA. Expression levels of Gli2 and bHLH factors were assessed using western blotting. NSCs proliferation was evaluated using CCK-8 assay, and neural differentiation was determined by immunofluorescence staining. Finally, the modified NSCs were transplanted into mice with SCI, and their effects were assessed using behavioral and histological tests. Our results demonstrated that Wnt3a promoted the expression of Mash1 through Gli2. Moreover, the expression of Ngn1 and Hes1 was up-regulated, while Hes5 was down-regulated. Wnt3a also promoted NSCs proliferation and neural differentiation through this signaling pathway. In vivo experiments showed that NSCs transplantation mediated by Wnt3a-Gli2 signaling increased the number of neurons and resulted in improved Basso Mouse Scale scores. In conclusion, our findings suggest that Gli2 plays a role in mediating the regulation of Wnt3a signaling on promoting NSCs proliferation and neural differentiation. This pathway is therefore important in NSCs-mediated SCI recovery.

Transcriptomic and Metabolomic Investigation on Leaf Necrosis Induced by ZmWus2 Transient Overexpression in Nicotiana benthamiana.

WUSCHEL (WUS) is a crucial transcription factor in regulating plant stem cell development, and its expression can also improve genetic transformation. However, the ectopic expression of WUS always causes pleiotropic effects during genetic transformation, making it important to understand the regulatory mechanisms underlying these phenomena. In our study, we found that the transient expression of the maize WUS ortholog ZmWus2 caused severe leaf necrosis in Nicotiana benthamiana. We performed transcriptomic and non-target metabolomic analyses on tobacco leaves during healthy to wilted states after ZmWus2 transient overexpression. Transcriptomic analysis revealed that ZmWus2 transformation caused active metabolism of inositol trisphosphate and glycerol-3-phosphate, while also upregulating plant hormone signaling and downregulating photosystem and protein folding pathways. Metabolomic analysis mainly identified changes in the synthesis of phenylpropanoid compounds and various lipid classes, including steroid synthesis. In addition, transcription factors such as ethylene-responsive factors (ERFs), the basic helix-loop-helix (bHLH) factors, and MYBs were found to be regulated by ZmWus2. By integrating these findings, we developed a WUS regulatory model that includes plant hormone accumulation, stress responses, lipid remodeling, and leaf necrosis. Our study sheds light on the mechanisms underlying WUS ectopic expression causing leaf necrosis and may inform the development of future genetic transformation strategies.

Integrated genome-wide chromatin accessibility and expression profile identify key transcription factors involved in bud endodormancy break in tea plants

It is essential for tea plants (Camellia sinensis) to undergo bud endodormancy in order to survive winter and grow again the following spring. Molecular pathways underlying the release and break of endodormancy, however, remain unclear. Accessible chromatin regions play a crucial role in precise gene expression regulation. Here, we profiled the chromatin accessibility and transcriptional features of transcription factors (TFs) using ATAC- and RNA-seq technologies. We revealed dynamic changes during bud dormancy break in tea plants and detected open chromatin regions and TF binding sites. Numerous transcripts associated with open chromatin regions were found to exhibit dormant-preferential expression among TF-target genes. We focused on several crucial TF motifs, including MADS-box, AP2EREBP, bZIP, MYB, bHLH, E2FDP, REMB3, TCP, and G2-like factors, that were enriched in open chromatin regions at different budbreak stages, and predicted networks of TF-target genes. Then, we used qRT-PCR to investigate the expression patterns of budbreak-related genes.We found a set of differentially regulated peaks of MADS-box TFs and related genes enriched in plant hormone signal transduction pathway, photosynthesis, secondary metabolism, plant-pathogen interaction functions. Correlation analysis with the phytohormones abscisic acid and indole-3-acetic acid also provided details of the regulation network. Moreover, we identified the core TFs (SVP, CO, AP1, SOC, ABI, CBF, FRUITFULL, and ULTRAPETALA) of several pathways related to budbreak, providing the guideposts for future study.

PpIBH1-1 limits internode elongation of peach shoot in a dose-dependent manner

Peach [Prunus persica (L.) Batsch] annual shoots grow up quickly, which limits the lighting and ventilation of an orchard. Atypical bHLH proteins IBH1(INCREASED LEAF INCLINATION1 BINDING bHLH1) play substantial roles in regulating cell elongation and plant stature. In this study, three PpIBH1s (PpIBH1–1/−2/−3) were identified in peach genome and contain a conserved AS domain and a characteristic HLH domain. The transcript levels of three PpIBH1s positively correlated with internode length, which gradually increased from apex to base along the peach shoots. This positive correlation was further confirmed in apple and poplar shoots. And the PpIBH1s gene were highly expressed in the shoot tips collected from twelve dwarf peach cultivars (gid1c mutants). In tissue-specific expression analysis, PpIBH1–1 are more highly expressed in tissues at the growth-arrested stage than cell-elongating. Transgenic Arabidopsis lines showed that different plant heights depending on the dose of PpIBH1–1 transcripts. And the dwarfing PpIBH1–1 transgenic lines were caused by the shorted cell length. PpIBH1–1 interacted with two bHLH factors (PpACE2 and PpLP1). These results suggested that PpIBH1–1 probably prevents internode elongation of peach shoots in a dose-dependent manner. Our work provided a foundation for properly controlling the growth of annual peach branches.

Cell-specific effects of the sole C. elegans Daughterless/E protein homolog, HLH-2, on nervous system development.

Are there common mechanisms of neurogenesis used throughout an entire nervous system? We explored to what extent canonical proneural class I/II bHLH complexes are responsible for neurogenesis throughout the entire Caenorhabditis elegans nervous system. Distinct, lineage-specific proneural class II bHLH factors are generally thought to operate via interaction with a common, class I bHLH subunit, encoded by Daughterless in flies, the E proteins in vertebrates and HLH-2 in C. elegans. To eliminate function of all proneuronal class I/II bHLH complexes, we therefore genetically removed maternal and zygotic hlh-2 gene activity. We observed broad effects on neurogenesis, but still detected normal neurogenesis in many distinct neuron-producing lineages of the central and peripheral nervous system. Moreover, we found that hlh-2 selectively affects some aspects of neuron differentiation while leaving others unaffected. Although our studies confirm the function of proneuronal class I/II bHLH complexes in many different lineages throughout a nervous system, we conclude that their function is not universal, but rather restricted by lineage, cell type and components of differentiation programs affected.

Antagonistic control of seed dormancy in rice by two bHLH transcription factors.

Preharvest sprouting (PHS) due to lack of seed dormancy seriously threatens crop production worldwide. As a complex quantitative trait, breeding of crop cultivars with suitable seed dormancy is hindered by limited useful regulatory genes. Here by repeatable phenotypic characterization of fixed recombinant individuals, we report a quantitative genetic locus, Seed Dormancy 6 (SD6), from aus-type rice, encoding a basic helix-loop-helix (bHLH) transcription factor, which underlies the natural variation of seed dormancy. SD6 and another bHLH factor inducer of C-repeat binding factors expression 2 (ICE2) function antagonistically in controlling seed dormancy by directly regulating the ABA catabolism gene ABA8OX3, and indirectly regulating the ABA biosynthesis gene NCED2 via OsbHLH048, in a temperature-dependent manner. The weak-dormancy allele of SD6 is common in cultivated rice but undergoes negative selection in wild rice. Notably, by genome editing SD6 and its wheat homologs, we demonstrated that SD6 is a useful breeding target for alleviating PHS in cereals under field conditions.

CO interacts with JAZ repressors and bHLH subgroup IIId factors to negatively regulate jasmonate signaling in Arabidopsis seedlings.

CONSTANS (CO) is a master flowering-time regulator that integrates photoperiodic and circadian signals in Arabidopsis thaliana. CO is expressed in multiple tissues, including young leaves and seedling roots, but little is known about the roles and underlying mechanisms of CO in mediating physiological responses other than flowering. Here, we show that CO expression is responsive to jasmonate. CO negatively modulated jasmonate-imposed root-growth inhibition and anthocyanin accumulation. Seedlings from co mutants were more sensitive to jasmonate, whereas overexpression of CO resulted in plants with reduced sensitivity to jasmonate. Moreover, CO mediated the diurnal gating of several jasmonate-responsive genes under long-day conditions. We demonstrate that CO interacts with JASMONATE ZIM-DOMAIN (JAZ) repressors of jasmonate signaling. Genetic analyses indicated that CO functions in a CORONATINE INSENSITIVE1 (COI1)-dependent manner to modulate jasmonate responses. Furthermore, CO physically associated with the basic helix-loop-helix (bHLH) subgroup IIId transcription factors bHLH3 and bHLH17. CO acted cooperatively with bHLH17 in suppressing jasmonate signaling, but JAZ proteins interfered with their transcriptional functions and physical interaction. Collectively, our results reveal the crucial regulatory effects of CO on mediating jasmonate responses and explain the mechanism by which CO works together with JAZ and bHLH subgroup IIId factors to fine-tune jasmonate signaling.

The bHLH Transcription Factors in Neural Development and Therapeutic Applications for Neurodegenerative Diseases.

The development of functional neural circuits in the central nervous system (CNS) requires the production of sufficient numbers of various types of neurons and glial cells, such as astrocytes and oligodendrocytes, at the appropriate periods and regions. Hence, severe neuronal loss of the circuits can cause neurodegenerative diseases such as Huntington's disease (HD), Parkinson's disease (PD), Alzheimer's disease (AD), and Amyotrophic Lateral Sclerosis (ALS). Treatment of such neurodegenerative diseases caused by neuronal loss includes some strategies of cell therapy employing stem cells (such as neural progenitor cells (NPCs)) and gene therapy through cell fate conversion. In this report, we review how bHLH acts as a regulator in neuronal differentiation, reprogramming, and cell fate determination. Moreover, several different researchers are conducting studies to determine the importance of bHLH factors to direct neuronal and glial cell fate specification and differentiation. Therefore, we also investigated the limitations and future directions of conversion or transdifferentiation using bHLH factors.

TNFα-Induced Oxidative Stress and Mitochondrial Dysfunction Alter Hypothalamic Neurogenesis and Promote Appetite Versus Satiety Neuropeptide Expression in Mice.

Maternal obesity results in programmed offspring hyperphagia and obesity. The increased offspring food intake is due in part to the preferential differentiation of hypothalamic neuroprogenitor cells (NPCs) to orexigenic (AgRP) vs. anorexigenic (POMC) neurons. The altered neurogenesis may involve hypothalamic bHLH (basic helix–loop–helix) neuroregulatory factors (Hes1, Mash1, and Ngn3). Whilst the underlying mechanism remains unclear, it is known that mitochondrial function is critical for neurogenesis and is impacted by proinflammatory cytokines such as TNFα. Obesity is associated with the activation of inflammation and oxidative stress pathways. In obese pregnancies, increased levels of TNFα are seen in maternal and cord blood, indicating increased fetal exposure. As TNFα influences neurogenesis and mitochondrial function, we tested the effects of TNFα and reactive oxidative species (ROS) hydrogen peroxide (H2O2) on hypothalamic NPC cultures from newborn mice. TNFα treatment impaired NPC mitochondrial function, increased ROS production and NPC proliferation, and decreased the protein expression of proneurogenic Mash1/Ngn3. Consistent with this, AgRP protein expression was increased and POMC was decreased. Notably, treatment with H2O2 produced similar effects as TNFα and also reduced the protein expression of antioxidant SIRT1. The inhibition of STAT3/NFκB prevented the effects of TNFα, suggesting that TNFα mediates its effects on NPCs via mitochondrial-induced oxidative stress that involves both signaling pathways.

Hox genes collaborate with helix-loop-helix factor Grainyhead to promote neuroblast apoptosis along the anterior-posterior axis of the Drosophila larval central nervous system.

Hox genes code for a family of a homeodomain containing transcription factors that use TALE-HD containing factors Pbx/Exd and Meis/Homothorax to specify the development of the anterior-posterior axis of an organism. However, the absence of TALE-HD containing factors from specific tissues emphasizes the need to identify and validate new Hox cofactors. In Drosophila central nervous system, Hox executes segment-specific apoptosis of neural stem cells (neuroblasts) and neurons. In abdominal segments of larval central nervous system, Hox gene Abdominal-A mediates neuroblast apoptosis with the help of Extradenticle and bHLH factor Grainyhead using a 717-bp apoptotic enhancer. In this study, we show that this enhancer is critical for abdominal neuroblast apoptosis and relies on 2 separable set of DNA-binding motifs responsible for its initiation and maintenance. Our results also show that Abdominal-A and Grainyhead interact through their highly conserved DNA-binding domains, and the DNA-binding specificity of Abdominal-A-homeodomain is important for it to interact with Grainyhead and essential for it to execute neuroblast apoptosis in central nervous system. We also establish that Grainyhead is required for Hox-dependent neuroblast apoptosis in Labial and Sex Combs Reduced expressing regions of the central nervous system, and it can physically interact with all the Hox proteins in vitro. Our biochemical and functional data collectively support the idea that Grainyhead can function as a Hox cofactor and help them carry out their in vivo roles during development.

The Iron Deficiency-Regulated Small Protein Effector FEP3/IRON MAN1 Modulates Interaction of BRUTUS-LIKE1 With bHLH Subgroup IVc and POPEYE Transcription Factors.

In light of climate change and human population growth one of the most challenging tasks is to generate plants that are Fe-efficient, resilient to low Fe supply and Fe-biofortified. For such endeavors, it is crucial to understand the regulation of Fe acquisition and allocation in plants. One open question is how identified Fe-regulatory proteins comprising positive and negative regulators act together to steer Fe homeostasis. bHLH transcription factors (TFs) belonging to the subgroups IVb and IVc can initiate a bHLH cascade controlling the –Fe response in roots. In Arabidopsis thaliana, the –Fe-induced genes are sub-divided into several gene co-expression clusters controlled by different sets of TFs. Some of the co-expressed genes encode regulatory E3 ligase proteins BRUTUS (BTS)/BTS-LIKE (BTSL) and small proteins belonging to the group of FE UPTAKE-INDUCING PEPTIDE/IRON MAN (FEP/IMA). Recently, it was described that FEP1/IMA3 and FEP3/IMA1 proteins inhibit the repression of bHLH factors by BTS. We had postulated that –Fe-regulated co-expression clusters provide new information about regulatory protein interaction complexes. Here, we report a targeted yeast two-hybrid screen among 23 proteins of the –Fe response. This identified a novel protein interactome involving another E3 ligase, namely BTSL1, basic helix-loop-helix (bHLH) protein POPEYE (PYE) and transcription factors of the subgroup IVc as well as FEP3/IMA1. Because of the difficulty in stable BTSL1 protein expression in plant cells, we used a yeast two hybrid-based deletion mapping, homology modeling and molecular docking, to pinpoint interaction sites in BTSL1 and FEP3/IMA1. bHLH IVc TFs have similar residues at their C-terminus as FEP3/IMA1 interacting sites. FEP3/IMA1 attenuated interaction of BTSL1 and bHLH proteins in a yeast three-hybrid assay, in line with physiological data pointing to enhanced Fe acquisition and allocation in FEP3/IMA1 overexpression and btsl1 btsl2 mutant plants. Hence, exploiting –Fe-induced gene co-expression networks identified FEP3/IMA1 as a small effector protein that binds and inhibits the BTSL1 complex with PYE and bHLH subgroup IVc proteins. Structural analysis resolved interaction sites. This information helps improving models of Fe regulation and identifying novel targets for breeding of Fe-efficient crops.

Myt1l haploinsufficiency leads to obesity and multifaceted behavioral alterations in mice

BackgroundThe zinc finger domain containing transcription factor Myt1l is tightly associated with neuronal identity and is the only transcription factor known that is both neuron-specific and expressed in all neuronal subtypes. We identified Myt1l as a powerful reprogramming factor that, in combination with the proneural bHLH factor Ascl1, could induce neuronal fate in fibroblasts. Molecularly, we found it to repress many non-neuronal gene programs, explaining its supportive role to induce and safeguard neuronal identity in combination with proneural bHLH transcriptional activators. Moreover, human genetics studies found MYT1L mutations to cause intellectual disability and autism spectrum disorder often coupled with obesity.MethodsHere, we generated and characterized Myt1l-deficient mice. A comprehensive, longitudinal behavioral phenotyping approach was applied.ResultsMyt1l was necessary for survival beyond 24 h but not for overall histological brain organization. Myt1l heterozygous mice became increasingly overweight and exhibited multifaceted behavioral alterations. In mouse pups, Myt1l haploinsufficiency caused mild alterations in early socio-affective communication through ultrasonic vocalizations. In adulthood, Myt1l heterozygous mice displayed hyperactivity due to impaired habituation learning. Motor performance was reduced in Myt1l heterozygous mice despite intact motor learning, possibly due to muscular hypotonia. While anxiety-related behavior was reduced, acoustic startle reactivity was enhanced, in line with higher sensitivity to loud sound. Finally, Myt1l haploinsufficiency had a negative impact on contextual fear memory retrieval, while cued fear memory retrieval appeared to be intact.LimitationsIn future studies, additional phenotypes might be identified and a detailed characterization of direct reciprocal social interaction behavior might help to reveal effects of Myt1l haploinsufficiency on social behavior in juvenile and adult mice.ConclusionsBehavioral alterations in Myt1l haploinsufficient mice recapitulate several clinical phenotypes observed in humans carrying heterozygous MYT1L mutations and thus serve as an informative model of the human MYT1L syndrome.

Epigenome accessibility changes before and after activation reveal distinct and progressive differentiation for human memory T cell subsets

Abstract Memory T cells (MTC), an indispensable part of adaptive immune memory, have historically been categorized by the cell surface proteins CCR7 and CD45RA into specialized subtypes (TCM, TEM, and TEMRA), each with unique functions. However, the epigenetic characteristics that distinguish the MTC subsets as well as the gene regulatory networks governing each MTC subsets’ response to activation remain poorly understood. Here we define and categorize the transcriptional and epigenetic differences of MTC and their respective primary subsets found in human blood, both in a resting state and after ex-vivo stimulation. Resting TCM were found to be relatively more similar to naïve cells in both CD4 and CD8 lineages, while TEM exhibited greater numbers of differentially expressed genes (DEG) and alterations to chromatin accessibility. Differentially accessible regions (DAR) discerning memory subsets contained binding motifs for factors thought to regulate memory formation from the bZIP, T-box and HMG families, as well as sites for novel bHLH factors MSC and AHR that may function in MTC development. Examining the effect of stimulation on MTC gene expression and chromatin identified unique DAR and DEG modules, some of which may have initially been primed by previous activation events in naive progenitors. Primed DAR were correlated with augmented expression of important genes in MTC after stimulation, suggesting an epigenetic mechanism of regulation. Ultimately these results describe the accumulation of epigenetic alterations during primary activation and during memory development that distinguish MTC from naïve T cells, enable differentiation into distinct subsets, and influence how MTC respond to secondary activation. Supported by grants from NIH (RO1 AI113021,T32 GM0008490)

Early Deletion of Neurod1 Alters Neuronal Lineage Potential and Diminishes Neurogenesis in the Inner Ear.

Neuronal development in the inner ear is initiated by expression of the proneural basic Helix-Loop-Helix (bHLH) transcription factor Neurogenin1 that specifies neuronal precursors in the otocyst. The initial specification of the neuroblasts within the otic epithelium is followed by the expression of an additional bHLH factor, Neurod1. Although NEUROD1 is essential for inner ear neuronal development, the different aspects of the temporal and spatial requirements of NEUROD1 for the inner ear and, mainly, for auditory neuron development are not fully understood. In this study, using Foxg1Cre for the early elimination of Neurod1 in the mouse otocyst, we showed that Neurod1 deletion results in a massive reduction of differentiating neurons in the otic ganglion at E10.5, and in the diminished vestibular and rudimental spiral ganglia at E13.5. Attenuated neuronal development was associated with reduced and disorganized sensory epithelia, formation of ectopic hair cells, and the shortened cochlea in the inner ear. Central projections of inner ear neurons with conditional Neurod1 deletion are reduced, unsegregated, disorganized, and interconnecting the vestibular and auditory systems. In line with decreased afferent input from auditory neurons, the volume of cochlear nuclei was reduced by 60% in Neurod1 mutant mice. Finally, our data demonstrate that early elimination of Neurod1 affects the neuronal lineage potential and alters the generation of inner ear neurons and cochlear afferents with a profound effect on the first auditory nuclei, the cochlear nuclei.

Fantastic four: bHLH factors and the making of the pollen.

Fantastic four: bHLH factors and the making of the pollen.

R2R3-MYB Transcription Factor NtMYB330 Regulates Proanthocyanidin Biosynthesis and Seed Germination in Tobacco (Nicotiana tabacum L.).

Proanthocyanidins (PAs) are important phenolic compounds and PA biosynthesis is regulated by a ternary MBW complex consisting of a R2R3-MYB regulator, a bHLH factor and a WDR protein. In this study, a tobacco R2R3-MYB factor NtMYB330 was characterized as the PA-specific regulator in which the PA biosynthesis was promoted in the flowers of NtMYB330-overexpressing lines while decreased in the flowers of ntmyb330 mutants. NtMYB330 can interact with flavonoid-related bHLH partner NtAn1b and WDR protein NtAn11-1, and the NtMYB330-NtAn1b complex is required to achieve strong transcriptional activation of the PA-related structural genes NtDFR1, NtANS1, NtLAR1 and NtANR1. Our data reveal that NtMYB330 regulates PA biosynthesis in seeds and affects seed germination, in which NtMYB330-overexpressing lines showed higher PA accumulations in seed coats and inhibited germination, while ntmyb330 mutants had reduced seed coat PAs and improved germination. NtMYB330 affects seed germination possibly through two mechanisms: modulating seed coat PAs to affect coat-imposed dormancy. In addition, NtMYB330 regulates the expressions of abscisic acid (ABA) and gibberellin acid (GA) signaling-related genes, affecting ABA-GA crosstalk and seed germination. This study reveals that NtMYB330 specifically regulates PA biosynthesis via formation of the MBW complex in tobacco flowers and affects germination through adjustment of PA concentrations and ABA/GA signaling in tobacco seeds.