The proper timing of Atoh1 expression is pivotal for hair cell subtype differentiation and the establishment of inner ear function.

Atoh1 overexpression is essential for hair cell (HC) regeneration in the sensory epithelium of mammalian auditory and vestibular organs. However, Atoh1 overexpression alone cannot induce fully mature and functional HCs in the mammalian inner ear. In the current study, we investigated the effect of Atoh1 constitutive overexpression in native HCs by manipulating Atoh1 expression at different developmental stages. We demonstrated that constitutive overexpression of Atoh1 in native vestibular HCs did not affect cell survival but did impair vestibular function by interfering with the subtype differentiation of HCs and hair bundle development. In contrast, Atoh1 overexpression in cochlear HCs impeded their maturation, eventually leading to gradual HC loss in the cochlea and hearing dysfunction. Our study suggests that time-restricted Atoh1 expression is essential for the differentiation and survival of HCs in the inner ear, and this is pivotal for both hearing and vestibular function re-establishment through Atoh1 overexpression-induced HC regeneration strategies.

Radiation-induced bone loss in mice is ameliorated by inhibition of HIF-2α in skeletal progenitor cells.

Radiotherapy remains a common treatment modality for cancer despite skeletal complications. However, there are currently no effective treatments for radiation-induced bone loss, and the consequences of radiotherapy on skeletal progenitor cell (SPC) survival and function remain unclear. After radiation, leptin receptor-expressing cells, which include a population of SPCs, become localized to hypoxic regions of the bone and stabilize the transcription factor hypoxia-inducible factor-2α (HIF-2α), thus suggesting a role for HIF-2α in the skeletal response to radiation. Here, we conditionally knocked out HIF-2α in leptin receptor-expressing cells and their descendants in mice. Radiation therapy in littermate control mice reduced bone mass; however, HIF-2α conditional knockout mice maintained bone mass comparable to nonirradiated control animals. HIF-2α negatively regulated the number of SPCs, bone formation, and bone mineralization. To test whether blocking HIF-2α pharmacologically could reduce bone loss during radiation, we administered a selective HIF-2α inhibitor called PT2399 (a structural analog of which was recently FDA-approved) to wild-type mice before radiation exposure. Pharmacological inhibition of HIF-2α was sufficient to prevent radiation-induced bone loss in a single-limb irradiation mouse model. Given that ~90% of patients who receive a HIF-2α inhibitor develop anemia because of off-target effects, we developed a bone-targeting nanocarrier formulation to deliver the HIF-2α inhibitor to mouse bone, to increase on-target efficacy and reduce off-target toxicities. Nanocarrier-loaded PT2399 prevented radiation-induced bone loss in mice while reducing drug accumulation in the kidney. Targeted inhibition of HIF-2α may represent a therapeutic approach for protecting bone during radiation therapy.

Three distinct Atoh1 enhancers cooperate for sound receptor hair cell development.

Cochlear hair cells (HCs) in the inner ear are responsible for sound detection. For HC fate specification, the master transcription factor Atoh1 is both necessary and sufficient. Atoh1 expression is dynamic and tightly regulated during development, but the cis-regulatory elements mediating this regulation remain unresolved. Unexpectedly, we found that deleting the only recognized Atoh1 enhancer, defined here as Eh1, failed to impair HC development. By using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we discovered two additional Atoh1 enhancers: Eh2 and Eh3. Notably, Eh2 deletion was sufficient for impairing HC development, and concurrent deletion of Eh1 and Eh2 or all three enhancers resulted in nearly complete absence of HCs. Lastly, we showed that Atoh1 binds to all three enhancers, consistent with its autoregulatory function. Our findings reveal that the cooperative action of three distinct enhancers underpins effective Atoh1 regulation during HC development, indicating potential therapeutic approaches for HC regeneration.

Hypothesis: Emerging Roles for Aryl Hydrocarbon Receptor in Orchestrating CoV-2-Related Inflammation.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the pathogenic agent of Coronavirus-Induced Disease-2019 (COVID-19), a multi-organ syndrome which primarily targets the respiratory system. In this review, considering the large amount of data pointing out the role of the Aryl hydrocarbon Receptor (AhR) in the inflammatory response and in the modulation of innate and adaptive immunity, we describe some mechanisms that strongly suggest its involvement in the management of COVID-19's inflammatory framework. It regulates both the expression of Angiotensin Converting Enzyme-2 (ACE-2) and its stabilizing partner, the Broad neutral Amino acid Transporter 1 (B0AT1). It induces Indolamine 2,3 dioxygenase (IDO-1), the enzyme which, starting from Tryptophan (Trp), produces Kynurenine (Kyn, Beta-Anthraniloyl-L-Alanine). The accumulation of Kyn and the depletion of Trp arrest T cell growth and induce apoptosis, setting up an immune-tolerant condition, whereas AhR and interferon type I (IFN-I) build a mutual inhibitory loop that also involves NF-kB and limits the innate response. AhR/Kyn binding boosts the production of Interleukin-6 (IL-6), thus reinforcing the inflammatory state and counteracting the IDO-dependent immune tolerance in the later stage of COVID-19. Taken together, these data depict a framework where sufficient clues suggest the possible participation of AhR in the management of COVID-19 inflammation, thus indicating an additional therapeutic target for this disease.

SbbHLH85, a bHLH member, modulates resilience to salt stress by regulating root hair growth in sorghum.

bHLH family proteins play an important role in plant stress response. However, the molecular mechanism regulating the salt response of bHLH is largely unknown. This study aimed to investigate the function and regulating mechanism of the sweet sorghum SbbHLH85 during salt stress. The results showed that SbbHLH85 was different from its homologs in other species. Also, it was a new atypical bHLH transcription factor and a key gene for root development in sweet sorghum. The overexpression of SbbHLH85 resulted in significantly increased number and length of root hairs via ABA and auxin signaling pathways, increasing the absorption of Na+. Thus, SbbHLH85 plays a negative regulatory role in the salt tolerance of sorghum. We identified a potential interaction partner of SbbHLH85, which was phosphate transporter chaperone PHF1 and modulated the distribution of phosphate, through screening a yeast two-hybrid library. Both yeast two-hybrid and BiFC experiments confirmed the interaction between SbbHLH85 and PHF1. The overexpression of SbbHLH85 led to a decrease in the expression of PHF1 as well as the content of Pi. Based on these results, we suggested that the increase in the Na+ content and the decrease in the Pi content resulted in the salt sensitivity of transgenic sorghum.

Sex-specific pubertal and metabolic regulation of Kiss1 neurons via Nhlh2.

Hypothalamic Kiss1 neurons control gonadotropin-releasing hormone release through the secretion of kisspeptin. Kiss1 neurons serve as a nodal center that conveys essential regulatory cues for the attainment and maintenance of reproductive function. Despite this critical role, the mechanisms that control kisspeptin synthesis and release remain largely unknown. Using Drop-Seq data from the arcuate nucleus of adult mice and in situ hybridization, we identified Nescient Helix-Loop-Helix 2 (Nhlh2), a transcription factor of the basic helix-loop-helix family, to be enriched in Kiss1 neurons. JASPAR analysis revealed several binding sites for NHLH2 in the Kiss1 and Tac2 (neurokinin B) 5' regulatory regions. In vitro luciferase assays evidenced a robust stimulatory action of NHLH2 on human KISS1 and TAC3 promoters. The recruitment of NHLH2 to the KISS1 and TAC3 promoters was further confirmed through chromatin immunoprecipitation. In vivo conditional ablation of Nhlh2 from Kiss1 neurons using Kiss1Cre:Nhlh2fl/fl mice induced a male-specific delay in puberty onset, in line with a decrease in arcuate Kiss1 expression. Females retained normal reproductive function albeit with irregular estrous cycles. Further analysis of male Kiss1Cre:Nhlh2fl/fl mice revealed higher susceptibility to metabolic challenges in the release of luteinizing hormone and impaired response to leptin. Overall, in Kiss1 neurons, Nhlh2 contributes to the metabolic regulation of kisspeptin and NKB synthesis and release, with implications for the timing of puberty onset and regulation of fertility in male mice.

Photoprotection during iron deficiency is mediated by the bHLH transcription factors PYE and ILR3.

Iron (Fe) is an essential micronutrient whose availability is limiting in many soils. During Fe deficiency, plants alter the expression of many genes to increase Fe uptake, distribution, and utilization. In a genetic screen for suppressors of Fe sensitivity in the E3 ligase mutant bts-3, we isolated an allele of the bHLH transcription factor (TF) ILR3, ilr3-4 We identified a striking leaf bleaching phenotype in ilr3 mutants that was suppressed by limiting light intensity, indicating that ILR3 is required for phototolerance during Fe deficiency. Among its paralogs that are thought to be partially redundant, only ILR3 was required for phototolerance as well as repression of genes under Fe deficiency. A mutation in the gene-encoding PYE, a known transcriptional repressor under Fe deficiency, also caused leaf bleaching. We identified singlet oxygen as the accumulating reactive oxygen species (ROS) in ilr3-4 and pye, suggesting photosensitivity is due to a PSII defect resulting in ROS production. During Fe deficiency, ilr3-4 and pye chloroplasts retain normal ultrastructure and, unlike wild type (WT), contain stacked grana similar to Fe-sufficient plants. Additionally, we found that the D1 subunit of PSII is destabilized in WT during Fe deficiency but not in ilr3-4 and pye, suggesting that PSII repair is accelerated during Fe deficiency in an ILR3- and PYE-dependent manner. Collectively, our results indicate that ILR3 and PYE confer photoprotection during Fe deficiency to prevent the accumulation of singlet oxygen, potentially by promoting reduction of grana stacking to limit excitation and facilitate repair of the photosynthetic machinery.

S1P Stimulates Erythropoietin Production in Mouse Renal Interstitial Fibroblasts by S1P1 and S1P3 Receptor Activation and HIF-2α Stabilization.

Erythropoietin (Epo) is the critical hormone for erythropoiesis. In adults, Epo is mainly produced by a subset of interstitial fibroblasts in the kidney, with minor amounts being produced in the liver and the brain. In this study, we used the immortalized renal interstitial fibroblast cell line FAIK F3-5 to investigate the ability of the bioactive sphingolipid sphingosine 1-phosphate (S1P) to stimulate Epo production and to reveal the mechanism involved. Stimulation of cells with exogenous S1P under normoxic conditions (21% O2) led to a dose-dependent increase in Epo mRNA and protein levels and subsequent release of Epo into the medium. S1P also enhanced the stabilization of HIF-2α, a key transcription factor for Epo expression. S1P-stimulated Epo mRNA and protein expression was abolished by HIF-2α mRNA knockdown or by the HIF-2 inhibitor compound 2. Furthermore, the approved S1P receptor modulator FTY720, and its active form FTY720-phosphate, both exerted a similar effect on Epo expression as S1P. The effect of S1P on Epo was antagonized by the selective S1P1 and S1P3 antagonists NIBR-0213 and TY-52156, but not by the S1P2 antagonist JTE-013. Moreover, inhibitors of the classical MAPK/ERK, the p38-MAPK, and inhibitors of protein kinase (PK) C and D all blocked the effect of S1P on Epo expression. Finally, the S1P and FTY720 effects were recapitulated in the Epo-producing human neuroblastoma cell line Kelly, suggesting that S1P receptor-dependent Epo synthesis is of general relevance and not species-specific. In summary, these data suggest that, in renal interstitial fibroblasts, which are the primary source of plasma Epo, S1P1 and 3 receptor activation upregulates Epo under normoxic conditions. This may have a therapeutic impact on disease situations such as chronic kidney disease, where Epo production is impaired, causing anemia, but it may also have therapeutic value as Epo can mediate additional tissue-protective effects in various organs.

Conditional Mutation of Hand1 in the Mouse Placenta Disrupts Placental Vascular Development Resulting in Fetal Loss in Both Early and Late Pregnancy.

Congenital heart defects (CHD) affect approximately 1% of all live births, and often require complex surgeries at birth. We have previously demonstrated abnormal placental vascularization in human placentas from fetuses diagnosed with CHD. Hand1 has roles in both heart and placental development and is implicated in CHD development. We utilized two conditionally activated Hand1A126fs/+ murine mutant models to investigate the importance of cell-specific Hand1 on placental development in early (Nkx2-5Cre) and late (Cdh5Cre) pregnancy. Embryonic lethality occurred in Nkx2-5Cre/Hand1A126fs/+ embryos with marked fetal demise occurring after E10.5 due to a failure in placental labyrinth formation and therefore the inability to switch to hemotrophic nutrition or maintain sufficient oxygen transfer to the fetus. Labyrinthine vessels failed to develop appropriately and vessel density was significantly lower by day E12.5. In late pregnancy, the occurrence of Cdh5Cre+;Hand1A126fs/+ fetuses was reduced from 29% at E12.5 to 20% at E18.5 and remaining fetuses exhibited reduced fetal and placental weights, labyrinth vessel density and placenta angiogenic factor mRNA expression. Our results demonstrate for the first time the necessity of Hand1 in both establishment and remodeling of the exchange area beyond early pregnancy and in patterning vascularization of the placental labyrinth crucial for maintaining pregnancy and successful fetal growth.

EPAS1 (Endothelial PAS Domain Protein 1) Orchestrates Transactivation of Endothelial ICAM1 (Intercellular Adhesion Molecule 1) by Small Nucleolar RNA Host Gene 5 (SNHG5) to Promote Hypoxic Pulmonary Hypertension.

[Figure: see text].

Roles of NPAS2 in circadian rhythm and disease.

NPAS2, a circadian rhythm gene encoding the neuronal PAS domain protein 2 (NPAS2), has received widespread attention because of its complex functions in cells and diverse roles in disease progression, especially tumorigenesis. NPAS2 binds with DNA at E-box sequences and forms heterodimers with another circadian protein, brain and muscle ARNT-like protein 1 (BMAL1). Nucleotide variations of the NPAS2 gene have been shown to influence the overall survival and risk of death of cancer patients, and differential expression of NPAS2 has been linked to patient outcomes in breast cancer, lung cancer, non-Hodgkin's lymphoma, and other diseases. Here, we review the latest advances in our understanding of NPAS2 with the aim of drawing attention to its potential clinical applications and prospects.

Key Signaling Pathways Regulate the Development and Survival of Auditory Hair Cells.

The loss of auditory sensory hair cells (HCs) is the most common cause of sensorineural hearing loss (SNHL). As the main sound transmission structure in the cochlea, it is necessary to maintain the normal shape and survival of HCs. In this review, we described and summarized the signaling pathways that regulate the development and survival of auditory HCs in SNHL. The role of the mitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase/protein kinase B (PI3K/Akt), Notch/Wnt/Atoh1, calcium channels, and oxidative stress/reactive oxygen species (ROS) signaling pathways are the most relevant. The molecular interactions of these signaling pathways play an important role in the survival of HCs, which may provide a theoretical basis and possible therapeutic interventions for the treatment of hearing loss.

Characterization and functional analysis of LoUDT1, a bHLH transcription factor related to anther development in the lily oriental hybrid Siberia (Lilium spp.).

Lily (Lilium spp.), with its beautiful flower, is an important horticultural crop and a popular ornamental plant, but because the abundant pollen pollutes the flowers and surroundings, its use is restricted. To solve this problem, the mechanism of pollen development in lily needs to be analyzed. However, the complex and delicate process of anther development in lily remains largely unknown. In this study, LoUDT1, a bHLH transcription factor (TF), was isolated and identified in lily. LoUDT1 was closely related to OsUDT1 of Oryza sativa and AtDYT1 of Arabidopsis. It was localized in the cytoplasm and nucleus and showed no transcriptional activation in yeast cells. LoUDT1 interacted with another bHLH TF, LoAMS, and the interaction depended on their BIF domains. LoUDT1 and LoAMS were both expressed in the anthers but showed different expression patterns. LoUDT1 was continuously expressed during the entire development of anthers, whereas LoAMS was only highly expressed early in anther development. With overexpression of LoUDT1 in Arabidopsis, normal anther development was affected and defective pollens were produced, which caused partial male sterility of transgenic plants. These defects depended on the level of LoUDT1 accumulation. By contrast, with the appropriate expression of LoUDT1 in a dyt1-3 mutant, normal pollen grains were produced, showing partial fertility. Thus, LoUDT1 might be a key regulator of anther development in lily. By further increasing the understanding of anther development, the results of this study can provide a theoretical basis for the molecular breeding of pollen-free lilies.

A novel gene LbHLH from the halophyte Limonium bicolor enhances salt tolerance via reducing root hair development and enhancing osmotic resistance.

BACKGROUND: Identifying genes involved in salt tolerance in the recretohalophyte Limonium bicolor could facilitate the breeding of crops with enhanced salt tolerance. Here we cloned the previously uncharacterized gene LbHLH and explored its role in salt tolerance. RESULTS: The 2,067-bp open reading frame of LbHLH encodes a 688-amino-acid protein with a typical helix-loop-helix (HLH) domain. In situ hybridization showed that LbHLH is expressed in salt glands of L. bicolor. LbHLH localizes to the nucleus, and LbHLH is highly expressed during salt gland development and in response to NaCl treatment. To further explore its function, we heterologously expressed LbHLH in Arabidopsis thaliana under the 35S promoter. The overexpression lines showed significantly increased trichome number and reduced root hair number. LbHLH might interact with GLABRA1 to influence trichome and root hair development, as revealed by yeast two-hybrid analysis. The transgenic lines showed higher germination percentages and longer roots than the wild type under NaCl treatment. Analysis of seedlings grown on medium containing sorbitol with the same osmotic pressure as 100 mM NaCl demonstrated that overexpressing LbHLH enhanced osmotic resistance. CONCLUSION: These results indicate that LbHLH enhances salt tolerance by reducing root hair development and enhancing osmotic resistance under NaCl stress.

Guard cells control hypocotyl elongation through HXK1, HY5, and PIF4.

The hypocotyls of germinating seedlings elongate in a search for light to enable autotrophic sugar production. Upon exposure to light, photoreceptors that are activated by blue and red light halt elongation by preventing the degradation of the hypocotyl-elongation inhibitor HY5 and by inhibiting the activity of the elongation-promoting transcription factors PIFs. The question of how sugar affects hypocotyl elongation and which cell types stimulate and stop that elongation remains unresolved. We found that overexpression of a sugar sensor, Arabidopsis hexokinase 1 (HXK1), in guard cells promotes hypocotyl elongation under white and blue light through PIF4. Furthermore, expression of PIF4 in guard cells is sufficient to promote hypocotyl elongation in the light, while expression of HY5 in guard cells is sufficient to inhibit the elongation of the hy5 mutant and the elongation stimulated by HXK1. HY5 exits the guard cells and inhibits hypocotyl elongation, but is degraded in the dark. We also show that the inhibition of hypocotyl elongation by guard cells' HY5 involves auto-activation of HY5 expression in other tissues. It appears that guard cells are capable of coordinating hypocotyl elongation and that sugar and HXK1 have the opposite effect of light on hypocotyl elongation, converging at PIF4.

The role of HIF proteins in maintaining the metabolic health of the intervertebral disc.

The physiologically hypoxic intervertebral disc and cartilage rely on the hypoxia-inducible factor (HIF) family of transcription factors to mediate cellular responses to changes in oxygen tension. During homeostatic development, oxygen-dependent prolyl hydroxylases, circadian clock proteins and metabolic intermediates control the activities of HIF1 and HIF2 in these tissues. Mechanistically, HIF1 is the master regulator of glycolytic metabolism and cytosolic lactate levels. In addition, HIF1 regulates mitochondrial metabolism by promoting flux through the tricarboxylic acid cycle, inhibiting downsteam oxidative phosphorylation and controlling mitochondrial health through modulation of the mitophagic pathway. Accumulation of metabolic intermediates from HIF-dependent processes contribute to intracellular pH regulation in the disc and cartilage. Namely, to prevent changes in intracellular pH that could lead to cell death, HIF1 orchestrates a bicarbonate buffering system in the disc, controlled by carbonic anhydrase 9 (CA9) and CA12, sodium bicarbonate cotransporters and an intracellular H+/lactate efflux mechanism. In contrast to HIF1, the role of HIF2 remains elusive; in disorders of the disc and cartilage, its function has been linked to both anabolic and catabolic pathways. The current knowledge of hypoxic cell metabolism and regulation of HIF1 activity provides a strong basis for the development of future therapies designed to repair the degenerative disc.

Two types of bHLH transcription factor determine the competence of the pericycle for lateral root initiation.

The molecular basis of the competence of the pericycle cell to initiate lateral root primordium formation is totally unknown. Here, we report that in Arabidopsis, two types of basic helix-loop-helix (bHLH) transcription factors, named PERICYCLE FACTOR TYPE-A (PFA) proteins and PERICYCLE FACTOR TYPE-B (PFB) proteins, govern the competence of pericycle cells to initiate lateral root primordium formation. Overexpression of PFA genes confers hallmark pericycle characteristics, including specific marker gene expression and auxin-induced cell division, and multiple loss-of-function mutations in PFA genes or the repression of PFB target genes results in the loss of this specific pericycle function. PFA and PFB proteins physically interact and are under mutual- and self-regulation, forming a positive feedback loop. This study unveils the transcriptional regulatory system that determines pericycle participation in lateral root initiation.

The membrane-localized protein kinase MAP4K4/TOT3 regulates thermomorphogenesis.

Plants respond to mild warm temperature conditions by increased elongation growth of organs to enhance cooling capacity, in a process called thermomorphogenesis. To this date, the regulation of thermomorphogenesis has been exclusively shown to intersect with light signalling pathways. To identify regulators of thermomorphogenesis that are conserved in flowering plants, we map changes in protein phosphorylation in both dicots and monocots exposed to warm temperature. We identify MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASE4 (MAP4K4)/TARGET OF TEMPERATURE3 (TOT3) as a regulator of thermomorphogenesis that impinges on brassinosteroid signalling in Arabidopsis thaliana. In addition, we show that TOT3 plays a role in thermal response in wheat, a monocot crop. Altogether, the conserved thermal regulation by TOT3 expands our knowledge of thermomorphogenesis beyond the well-studied pathways and can contribute to ensuring food security under a changing climate.

Striatal Direct Pathway Targets Npas1+ Pallidal Neurons.

The classic basal ganglia circuit model asserts a complete segregation of the two striatal output pathways. Empirical data argue that, in addition to indirect-pathway striatal projection neurons (iSPNs), direct-pathway striatal projection neurons (dSPNs) innervate the external globus pallidus (GPe). However, the functions of the latter were not known. In this study, we interrogated the organization principles of striatopallidal projections and their roles in full-body movement in mice (both males and females). In contrast to the canonical motor-promoting response of dSPNs in the dorsomedial striatum (DMSdSPNs), optogenetic stimulation of dSPNs in the dorsolateral striatum (DLSdSPNs) suppressed locomotion. Circuit analyses revealed that dSPNs selectively target Npas1+ neurons in the GPe. In a chronic 6-hydroxydopamine lesion model of Parkinson's disease, the dSPN-Npas1+ projection was dramatically strengthened. As DLSdSPN-Npas1+ projection suppresses movement, the enhancement of this projection represents a circuit mechanism for the hypokinetic symptoms of Parkinson's disease that has not been previously considered. In sum, our results suggest that dSPN input to the GPe is a critical circuit component that is involved in the regulation of movement in both healthy and parkinsonian states.SIGNIFICANCE STATEMENT In the classic basal ganglia model, the striatum is described as a divergent structure: it controls motor and adaptive functions through two segregated, opposing output streams. However, the experimental results that show the projection from direct-pathway neurons to the external pallidum have been largely ignored. Here, we showed that this striatopallidal subpathway targets a select subset of neurons in the external pallidum and is motor-suppressing. We found that this subpathway undergoes changes in a Parkinson's disease model. In particular, our results suggest that the increase in strength of this subpathway contributes to the slowness or reduced movements observed in Parkinson's disease.