Revisiting the Potency of Tbx2 Expression in Transforming Outer Hair Cells into Inner Hair Cells at Multiple Ages In Vivo.

The mouse auditory organ cochlea contains two types of sound receptors: inner hair cells (IHCs) and outer hair cells (OHCs). Tbx2 is expressed in IHCs but repressed in OHCs, and neonatal OHCs that misexpress Tbx2 transdifferentiate into IHC-like cells. However, the extent of this switch from OHCs to IHC-like cells and the underlying molecular mechanism remain poorly understood. Furthermore, whether Tbx2 can transform fully mature adult OHCs into IHC-like cells is unknown. Here, our single-cell transcriptomic analysis revealed that in neonatal OHCs misexpressing Tbx2, 85.6% of IHC genes, including Slc17a8, are upregulated, but only 38.6% of OHC genes, including Ikzf2 and Slc26a5, are downregulated. This suggests that Tbx2 cannot fully reprogram neonatal OHCs into IHCs. Moreover, Tbx2 also failed to completely reprogram cochlear progenitors into IHCs. Lastly, restoring Ikzf2 expression alleviated the abnormalities detected in Tbx2+ OHCs, which supports the notion that Ikzf2 repression by Tbx2 contributes to the transdifferentiation of OHCs into IHC-like cells. Our study evaluates the effects of ectopic Tbx2 expression on OHC lineage development at distinct stages of either male or female mice and provides molecular insights into how Tbx2 disrupts the gene expression profile of OHCs. This research also lays the groundwork for future studies on OHC regeneration.

In situ regeneration of inner hair cells in the damaged cochlea by temporally regulated co-expression of Atoh1 and Tbx2.

Cochlear inner hair cells (IHCs) are primary sound receptors, and are therefore a target for developing treatments for hearing impairment. IHC regeneration in vivo has been widely attempted, although not yet in the IHC-damaged cochlea. Moreover, the extent to which new IHCs resemble wild-type IHCs remains unclear, as is the ability of new IHCs to improve hearing. Here, we have developed an in vivo mouse model wherein wild-type IHCs were pre-damaged and nonsensory supporting cells were transformed into IHCs by ectopically expressing Atoh1 transiently and Tbx2 permanently. Notably, the new IHCs expressed the functional marker vGlut3 and presented similar transcriptomic and electrophysiological properties to wild-type IHCs. Furthermore, the formation efficiency and maturity of new IHCs were higher than those previously reported, although marked hearing improvement was not achieved, at least partly due to defective mechanoelectrical transduction (MET) in new IHCs. Thus, we have successfully regenerated new IHCs resembling wild-type IHCs in many respects in the damaged cochlea. Our findings suggest that the defective MET is a critical barrier that prevents the restoration of hearing capacity and should thus facilitate future IHC regeneration studies.

Free-Breathing Liver Magnetic Resonance Imaging With Respiratory Frequency-Modulated Continuous-Wave Radar-Trigger Technique: A Preliminary Study.

Purpose: The aim of this study is to evaluate the performance of free-breathing liver MRI with a novel respiratory frequency-modulated continuous-wave radar-trigger (FT) technique on T2-weighted imaging (T2WI) and diffusion-weighted imaging (DWI) for both healthy volunteers and patients in comparison to navigator-trigger (NT) and belt-trigger (BT) techniques. Methods: In this prospective study, 17 healthy volunteers and 23 patients with known or suspected liver diseases were enrolled. Six sequences (T2WI and DWI with FT, NT, and BT techniques) were performed in each subject. Quantitative evaluation and qualitative assessment were analyzed by two radiologists. Overall image quality, blurring, motion artifacts, and liver edge delineations were rated on a 4-point Likert scale. The liver and lesion signal-to-noise ratio (SNR), the lesion-to-liver contrast-to-noise ratio (CNR), as well as the apparent diffusion coefficient (ADC) value were quantitatively calculated. Results: For volunteers, there were no significant differences in the image quality Likert scores and quantitative parameters on T2WI and DWI with three respiratory-trigger techniques. For patients, NT was superior to other techniques for image quality on T2WI; conversely, little difference was found on DWI in qualitative assessment. The mean SNR of the liver on T2WI and DWI with BT, NT, and FT techniques was similar in patients, which is in line with volunteers. FT performed better in terms of higher SNR (705.13 ± 434.80) and higher CNR (504.41 ± 400.69) on DWI at b50 compared with BT (SNR: 651.83 ± 401.16; CNR:429.24 ± 404.11) and NT (SNR: 639.41 ± 407.98; CNR: 420.64 ± 416.61) (p < 0.05). The mean ADC values of the liver and lesion with different techniques in both volunteers and patients showed non-significant difference. Conclusion: For volunteers, the performance of T2WI as well as DWI with three respiratory-trigger techniques was similarly good. As for patients, FT-DWI is superior to BT and NT techniques in terms of higher lesion SNR and CNR at b50.

Development and transdifferentiation into inner hair cells require Tbx2.

Atoh1 is essential for the development of both outer hair cells (OHCs) and inner hair cells (IHCs) in the mammalian cochlea. Whereas Ikzf2 is necessary for OHC development, the key gene required for IHC development remains unknown. We found that deletion of Tbx2 in neonatal IHCs led to their transdifferentiation into OHCs by repressing 26.7% of IHC genes and inducing 56.3% of OHC genes, including Ikzf2. More importantly, persistent expression of Tbx2 coupled with transient Atoh1 expression effectively reprogrammed non-sensory supporting cells into new IHCs expressing the functional IHC marker vGlut3. The differentiation status of these new IHCs was considerably more advanced than that previously reported. Thus, Tbx2 is essential for IHC development and co-upregulation of Tbx2 with Atoh1 in supporting cells represents a new approach for treating deafness related to IHC degeneration.

In vivo ectopic Ngn1 and Neurod1 convert neonatal cochlear glial cells into spiral ganglion neurons.

Damage or degeneration of inner ear spiral ganglion neurons (SGNs) causes hearing impairment. Previous in vitro studies indicate that cochlear glial cells can be reprogrammed into SGNs, however, it remains unknown whether this can occur in vivo. Here, we show that neonatal glial cells can be converted, in vivo, into SGNs (defined as new SGNs) by simultaneous induction of Neurog1 (Ngn1) and Neurod1. New SGNs express SGN markers, Tuj1, Map2, Prox1, Mafb and Gata3, and reduce glial cell marker Sox10 and Scn7a. The heterogeneity within new SGNs is illustrated by immunostaining and transcriptomic assays. Transcriptomes analysis indicates that well reprogrammed SGNs are similar to type I SGNs. In addition, reprogramming efficiency is positively correlated with the dosage of Ngn1 and Neurod1, but declined with aging. Taken together, our in vivo data demonstrates the plasticity of cochlear neonatal glial cells and the capacity of Ngn1 and Neurod1 to reprogram glial cells into SGNs. Looking ahead, we expect that combination of Neurog1 and Neurod1 along with other factors will further boost the percentage of fully converted (Mafb+/Gata3+) new SGNs.

Comprehensive transcriptome analysis of cochlear spiral ganglion neurons at multiple ages.

Inner ear cochlear spiral ganglion neurons (SGNs) transmit sound information to the brainstem. Recent single cell RNA-Seq studies have revealed heterogeneities within SGNs. Nonetheless, much remains unknown about the transcriptome of SGNs, especially which genes are specifically expressed in SGNs. To address these questions, we needed a deeper and broader gene coverage than that in previous studies. We performed bulk RNA-Seq on mouse SGNs at five ages, and on two reference cell types (hair cells and glia). Their transcriptome comparison identified genes previously unknown to be specifically expressed in SGNs. To validate our dataset and provide useful genetic tools for this research field, we generated two knockin mouse strains: Scrt2-P2A-tdTomato and Celf4-3xHA-P2A-iCreER-T2A-EGFP. Our comprehensive analysis confirmed the SGN-selective expression of the candidate genes, testifying to the quality of our transcriptome data. These two mouse strains can be used to temporally label SGNs or to sort them.

Cloning and functional analysis of five TERMINAL FLOWER 1/CENTRORADIALIS-like genes from Hevea brasiliensis.

Five TERMINAL FLOWER 1 (TFL1)/CENTRORADIALIS (CEN)-like genes were isolated and characterized from rubber tree (Hevea brasiliensis). All genes, except HbCEN1, were found to have conserved genomic organization, characteristic of the phosphatidyl ethanolamine-binding protein (PEBP) family. Overexpression of all of them delayed flowering and altered flower architecture compared with the wild-type (wt) counterpart. In addition, as premature-flowering of the terminal bud was successfully overcome in the tfl1-1 mutant of Arabidopsis, all these genes have a potential function similar to TFL1. Quantitative reverse transcriptase-polymerase chain reaction analysis showed higher expressions of HbCEN1 and HbCEN2 in the shoot apices and stems of both immature and mature rubber trees than in reproductive organs. HbTFL1-1 and HbTFL1-2 expression was confined to roots of 3-month-old seedlings and HbTFL1-3 was significantly higher in the shoot apices of these seedlings. These results suggested that HbCEN1 and HbCEN2 could be associated with the development of vegetative growth, whereas HbTFL1-1, HbTFL1-2 and HbTFL1-3 seem to be mainly related with maintenance of juvenility. In addition, four of the five genes displayed variable diurnal expression, HbTFL1-1 and HbTFL1-3 being mainly expressed during the night whereas HbCEN1 and HbCEN2 showed irregular diurnal rhythms.

Identification, Functional Study, and Promoter Analysis of HbMFT1, a Homolog of MFT from Rubber Tree (Hevea brasiliensis).

A homolog of MOTHER OF FT AND TFL1 (MFT) was isolated from Hevea brasiliensis and its biological function was investigated. Protein multiple sequence alignment and phylogenetic analysis revealed that HbMFT1 conserved critical amino acid residues to distinguish MFT, FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1)-like proteins and showed a closer genetic relationship to the MFT-like group. The accumulation of HbMFT1 was generally detected in various tissues except pericarps, with the highest expression in embryos and relatively higher expression in roots and stems of seedlings, flowering inflorescences, and male and female flowers. HbMFT1 putative promoter analysis showed that tissue-specific, environmental change responsive and hormone-signaling responsive elements were generally present. HbMFT1 was strongly induced under a short-day condition at 28 °C, with the highest expression after the onset of a day. Overexpression of HbMFT1 inhibited seed germination, seedling growth, and flowering in transgenic Arabidopsis. The qRT-PCR further confirmed that APETALA1 (AP1) and FRUITFULL (FUL) were drastically down-regulated in 35S::HbMFT1 plants. A histochemical ß-glucuronidase (GUS) assay showed that HbMFT1::GUS activity was mainly detected in stamens and mature seeds coinciding with its original expression and notably induced in rosette leaves and seedlings of transgenic Arabidopsis by exogenous abscisic acid (ABA) due to the presence of ABA cis-elements in HbMFT1 promoter. These results suggested that HbMFT1 was mainly involved in maintenance of seed maturation and stamen development, but negatively controlled germination, growth and development of seedlings and flowering. In addition, the HbMFT1 promoter can be utilized in controlling transgene expression in stamens and seeds of rubber tree or other plant species.

Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display.

Powdery mildew is an important disease of rubber trees caused by Oidium heveae B. A. Steinmann. As far as we know, none of the resistance genes related to powdery mildew have been isolated from the rubber tree. There is little information available at the molecular level regarding how a rubber tree develops defense mechanisms against this pathogen. We have studied rubber tree mRNA transcripts from the resistant RRIC52 cultivar by differential display analysis. Leaves inoculated with the spores of O. heveae were collected from 0 to 120 hpi in order to identify pathogen-regulated genes at different infection stages. We identified 78 rubber tree genes that were differentially expressed during the plant-pathogen interaction. BLAST analysis for these 78 ESTs classified them into seven functional groups: cell wall and membrane pathways, transcription factor and regulatory proteins, transporters, signal transduction, phytoalexin biosynthesis, other metabolism functions, and unknown functions. The gene expression for eight of these genes was validated by qRT-PCR in both RRIC52 and the partially susceptible Reyan 7-33-97 cultivars, revealing the similar or differential changes of gene expressions between these two cultivars. This study has improved our overall understanding of the molecular mechanisms of rubber tree resistance to powdery mildew.

Shedding of c-Met ectodomain correlates with c-Met expression in non-small cell lung cancer.

OBJECTIVE: The aim of this study is to reveal the correlation of shedding and expression of c-Met in non-small cell lung cancer (NSCLC) patient. MATERIALS AND METHODS: We measured soluble c-Met and c-Met level in a panel of pre-clinical models and 197 advanced Chinese NSCLC patients by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. RESULTS: Shedding of soluble c-Met associates with total c-Met amount in pre-clinical models, and soluble c-Met correlates with both c-Met expression level and tumor size in human, high soluble c-Met predicts poorer outcome.