Genomic, transcriptomic, and metabolomic analyses provide insights into the evolution and development of a medicinal plant Saposhnikovia divaricata (Apiaceae).

Saposhnikovia divaricata, 2n = 2x = 16, as a perennial species, is widely distributed in China, Mongolia, Russia, etc. It is a traditional Chinese herb used to treat tetanus, rubella pruritus, rheumatic arthralgia, and other diseases. Here, we assembled a 2.07 Gb and N50 scaffold length of 227.67 Mb high-quality chromosome-level genome of S. divaricata based on the PacBio Sequel II sequencing platform. The total number of genes identified was 42 948, and 42 456 of them were functionally annotated. A total of 85.07% of the genome was composed of repeat sequences, comprised mainly of long terminal repeats (LTRs) which represented 73.7% of the genome sequence. The genome size may have been affected by a recent whole-genome duplication event. Transcriptional and metabolic analyses revealed bolting and non-bolting S. divaricata differed in flavonoids, plant hormones, and some pharmacologically active components. The analysis of its genome, transcriptome, and metabolome helped to provide insights into the evolution of bolting and non-bolting phenotypes in wild and cultivated S. divaricata and lays the basis for genetic improvement of the species.

Ganoderma lucidum Polysaccharides Ameliorate Acetaminophen-Induced Acute Liver Injury by Inhibiting Oxidative Stress and Apoptosis along the Nrf2 Pathway.

The excessive employment of acetaminophen (APAP) is capable of generating oxidative stress and apoptosis, which ultimately result in acute liver injury (ALI). Ganoderma lucidum polysaccharides (GLPs) exhibit hepatoprotective activity, yet the protective impact and potential mechanism of GLPs in relation to APAP-induced ALI remain ambiguous. The intention of this research was to scrutinize the effect of GLPs on APAP-induced ALI and to shed light on their potential mechanism. The results demonstrated that GLPs were capable of notably alleviating the oxidative stress triggered by APAP, as shown through a significant drop in the liver index, the activities of serum ALT and AST, and the amounts of ROS and MDA in liver tissue, along with an increase in the levels of SOD, GSH, and GSH-Px. Within these, the hepatoprotective activity at the high dose was the most conspicuous, and its therapeutic efficacy surpassed that of the positive drug (bifendate). The results of histopathological staining (HE) and apoptosis staining (TUNEL) indicated that GLPs could remarkably inhibit the necrosis of hepatocytes, the permeation of inflammatory cells, and the occurrence of apoptosis induced by APAP. Moreover, Western blot analysis manifested that GLPs enhanced the manifestation of Nrf2 and its subsequent HO-1, GCLC, and NQO1 proteins within the Nrf2 pathway. The results of qPCR also indicated that GLPs augmented the expression of antioxidant genes Nrf2, HO-1, GCLC, and NQO1. The results reveal that GLPs are able to set off the Nrf2 signaling path and attenuate ALI-related oxidative stress and apoptosis, which is a potential natural medicine for the therapy of APAP-induced liver injury.

Advances in Phytochemistry and Modern Pharmacology of Saposhnikovia Divaricata (Turcz.) Schischk.

Saposhnikovia divaricata (Turcz.) Schischk (S. divaricata, Fangfeng) is a herb in the Apiaceae family, and its root has been used since the Western Han Dynasty (202 B.C.). Chromones and coumarins are the pharmacologically active substances in S. divaricata. Modern phytochemical and pharmacological studies have demonstrated their antipyretic, analgesic, anti-inflammatory, antioxidant, anti-tumor, and anticoagulant activities. Technological and analytical strategy theory advancements have yielded novel results; however, most investigations have been limited to the main active substances-chromones and coumarins. Hence, we reviewed studies related to the chemical composition and pharmacological activity of S. divaricata, analyzed the developing trends and challenges, and proposed that research should focus on components' synergistic effects. We also suggested that, the structure-effect relationship should be prioritized in advanced research.

Adenovirus-mediated effects of Wnt and Notch signalling pathways on hair cell regeneration in mice.

Some genes are delivered to cochleae by adenoviruses to restore partial hearing function. This provides promising prospects for gene therapies for hearing loss from hair cell damage. To study the adenovirus (AD)-mediated effect of the Wnt and Notch signalling pathways on hair cell regeneration in the mouse cochlea, we constructed a ß-catenin-adenovirus (ß-catenin-AD) to increase the activity of the Wnt signalling pathway and a NICD (intracellular domain of Notch1)-RNAi-adenovirus to decrease the activity of the Notch signalling pathway (NICD-RNAi-AD). Our study indicated that approximately 40% of supporting cells in the cochleae damaged by gentamicin were infected with the adenoviruses. Following the ß-catenin-AD-mediated increase in Wnt signalling pathway activity, mitotic regeneration was increased, while direct transdifferentiation was increased after the NICD-RNAi-AD-mediated decrease in Notch signalling pathway activity. The expected synergistic interaction on hair cell regeneration was not obtained after coinfection of ß-catenin-AD and NICD-RNAi-AD into the damaged cochleae, which might be due to the low cotransfection efficiency to supporting cells. Our study indicated that it may be possible to develop AD mediated gene therapies for hearing loss that act by regulating the Wnt and Notch signalling pathways.

Conditioned media of deer antler stem cells accelerate regeneration of alveolar bone defects in rats.

The destruction of periodontal alveolar bone (AB) caused by periodontitis is regarded as one of the major reasons for tooth loss. The inhibition of bone resorption and regeneration of lost AB are the desirable outcomes in clinical practice but remain in challenge. The use of mesenchymal stem cells (MSCs) is one current approach for achieving true restoration of AB defects (ABD). Antler stem cells (AnSC) are capable of renewing a huge mammalian bony appendage, the deer antler, suggesting an unparalleled potential for bone regeneration. Herein, we investigated the effectiveness of deer AnSCs conditioned medium (CM, AnSC-CM) for repair of surgically-created ABD using a rat model and sought to define the underlying mechanisms. The results showed that AnSC-CM effectively induced regeneration of AB tissue; the outcome was significantly better than human bone marrow mesenchymal stem cell conditioned medium (hBMSC-CM). AnSC-CM treatment upregulated osteogenic factors and downregulated osteoclastic differentiation factors; stimulated proliferation, migration and differentiation of resident MSCs toward osteogenic lineage cells; modulated macrophage polarization toward the M2 phenotype and suppressed osteoclastogenesis. That AnSC-CM resulted in better outcomes than hBMSC-CM in treating ABD was attributed to the cell compatibility as both AnSCs and AB tissue are neural crest-derived. In conclusion, the effects of AnSC-CM on AB tissue regeneration were achieved through both promotion of osteogenesis and inhibition of osteoclastogenesis. We believe that AnSC-CM is a candidate for effective treatment of ABD in dental clinical practice but will require investment in further development.

Characterization of the complete chloroplast genome of Clematis orientalis (Ranunculaceae).

The medicinal plant Clematis orientalis L. belongs to the family Ranunculaceae. In this study, we determined the complete chloroplast genome sequence of C. orientalis and its phylogenetic relationships with other species. The complete chloroplast genome of C. glauca is 159,518 bp in length, circular in structure, and has four regions including a large single-copy (LSC) region of 79,453 bp; a small single-copy (SSC) region of 17,997 bp; and two inverted repeat (IR) regions of 31,034 bp. The GC content of the genome is 38%, and those of LSC, SSC, and IR regions are 36.2, 31.4, and 42%, respectively. The genome encodes 129 unique genes, including 85 protein-coding genes, 36 tRNA genes, and eight rRNA genes. Phylogenomic analysis reveals that C. orientalis is most closely related to C. aethusifolia. This study contributes to better understanding of phylogenetic relationships of Ranunculaceae.

The complete chloroplast genome of Clematis hexapetala (Ranunculaceae) and its phylogenetic analysis.

Clematis hexapetala Pall. (1776) is a traditional Chinese medicine belonging to the Ranunculaceae. In this study, the complete chloroplast genome was sequenced through Illumina platform, cp was circular DNA molecule of 159,538 bp in length with a typical quadripartite structure, consisting of four regions: two copies of inverted repeat region (IRs: 31,039 bp), a large single-copy (LSC: 79,333 bp) region, a small single-copy (SSC: 18,127 bp) region. The chloroplast genome encodes a total of 135 genes, including 91 CDS genes, 36 tRNA genes, 8 rRNA genes. Phylogenetic analysis based on complete genes shows that C. hexapetala closely related to C. taeguensis in the genus Clematis. This study improves our comprehension of the chloroplast genome and its phylogenetic relationships within Ranunculaceae.

Enhancing Text Generation via Parse Tree Embedding.

Natural language generation (NLG) is a core component of machine translation, dialogue systems, speech recognition, summarization, and so forth. The existing text generation methods tend to be based on recurrent neural language models (NLMs), which generate sentences from encoding vector. However, most of these models lack explicit structured representation for text generation. In this work, we introduce a new generative model for NLG, called Tree-VAE. First it samples a sentence from the training corpus and then generates a new sentence based on the corresponding parse tree embedding vector. Tree-LSTM is used in collaboration with the Stanford Parser to retrieve sentence construction data, which is then used to train a conditional discretization autoencoder generator based on the embeddings of sentence patterns. The proposed model is extensively evaluated on three different datasets. The experimental results proved that the proposed model can generate substantially more diverse and coherent text than existing baseline methods.

Complete chloroplast genome characterization and phylogenetic analysis of Clematis mandshurica (Ranunculaceae).

The complete chloroplast genome sequence of Clematis mandshurica Ruprecht (1867), a specie of the Ranunculaceae family, and its phylogenetic relationships with other species have been reported in this study. The complete chloroplast genome of C. mandshurica is 159,563 bp in length, including a large single-copy (LSC) region of 79,360 bp, a small single-copy (SSC) region of 18,121 bp, and a pair of identical inverted repeat regions (IRs) of 31,041 bp. The genome encodes a total of 132 genes, including 90 protein-coding genes, 34 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. The phylogenetic analysis reveals that C. mandshurica was found to be closest to Clematis taeguensis. The complete chloroplast genome of C. mandshurica contributes to a better understanding of phylogenetic relationships among Clematis species.

Assembly of yield heterosis of an elite rice hybrid is promising by manipulating dominant quantitative trait loci.

In the past, rice hybrids with strong heterosis have been obtained empirically, by developing and testing thousands of combinations. Here, we aimed to determine whether heterosis of an elite hybrid could be achieved by manipulating major quantitative trait loci. We used 202 chromosome segment substitution lines from the elite hybrid Shanyou 63 to evaluate single segment heterosis (SSH) of yield per plant and identify heterotic loci. All nine detected heterotic loci acted in a dominant fashion, and no SSH exhibited overdominance. Functional alleles of key yield-related genes Ghd7, Ghd7.1, Hd1, and GS3 were dispersed in both parents. No functional alleles of three investigated genes were expressed at higher levels in the hybrids than in the more desirable parents. A hybrid pyramiding eight heterotic loci in the female parent Zhenshan 97 background had a comparable yield to Shanyou 63 and much higher yield than Zhenshan 97. Five hybrids pyramiding eight or nine heterotic loci in the combined parental genome background showed similar yield performance to that of Shanyou 63. These results suggest that dominance underlying functional complementation is an important contributor to yield heterosis and that heterosis assembly might be successfully promised by manipulating several major dominant heterotic loci.

Foliar nutrient allocation patterns in Banksia attenuata and Banksia sessilis differing in growth rate and adaptation to low-phosphorus habitats.

BACKGROUND AND AIMS: Phosphorus (P) and nitrogen (N) are essential nutrients that frequently limit primary productivity in terrestrial ecosystems. Efficient use of these nutrients is important for plants growing in nutrient-poor environments. Plants generally reduce foliar P concentration in response to low soil P availability. We aimed to assess ecophysiological mechanisms and adaptive strategies for efficient use of P in Banksia attenuata (Proteaceae), naturally occurring on deep sand, and B. sessilis, occurring on shallow sand over laterite or limestone, by comparing the allocation of P among foliar P fractions. METHODS: We carried out pot experiments with slow-growing B. attenuata, which resprouts after fire, and faster growing opportunistic B. sessilis, which is killed by fire, on substrates with different P availability using a randomized complete block design. We measured leaf P and N concentrations, photosynthesis, leaf mass per area, relative growth rate and P allocated to major biochemical fractions in B. attenuata and B. sessilis. KEY RESULTS: The two species had similarly low foliar total P concentrations, but distinct patterns of P allocation to P-containing fractions. The foliar total N concentration of B. sessilis was greater than that of B. attenuata on all substrates. The foliar total P and N concentrations in both species decreased with decreasing P availability. The relative growth rate of both species was positively correlated with concentrations of both foliar nucleic acid P and total N, but there was no correlation with other P fractions. Faster growing B. sessilis allocated more P to nucleic acids than B. attenuata did, but other fractions were similar. CONCLUSIONS: The nutrient allocation patterns in faster growing opportunistic B. sessilis and slower growing B. attenuata revealed different strategies in response to soil P availability which matched their contrasting growth strategy.

Signaling pathways (Notch, Wnt, Bmp and Fgf) have additive effects on hair cell regeneration in the chick basilar papilla after streptomycin injury in vitro: Additive effects of signaling pathways on hair cell regeneration.

Hair cells can be regenerated after damage by transdifferentiation in which a supporting cell directly differentiates into a hair cell without mitosis. However, such regeneration is at the cost of exhausting the support cells in the mammalian mature cochlea. Thus, more effective methods should be found to promote mitotic regeneration but partially preserve support cells after damage. To address the issue, we first injured hair cells in the chick basilar papillae (BP) by treatment with streptomycin in vitro. We then compared the mitotic regeneration on the neural side in the middle part of BP after treatment with a pharmacological inhibitor or agonist of the Notch (DAPT), Wnt (LiCl), Bmp (Noggin) or Fgf (SU5402) signaling pathway, with that after treatment with combinations of two or three inhibitors or agonist of these pathways. Our results indicate that treatments with a single inhibitor or agonist of the Notch, Wnt, Bmp or Fgf signaling pathway could significantly increase mitotic regeneration as well as direct transdifferentiation. The results also show that hair cells (Myosin 7a+), support cells (Sox2+) and mitotically regenerated hair cells (Myosin 7a+/Sox2+/BrdU+) increased significantly on the neural side in the middle part of BP after two or three combinations of the inhibition of Notch, Bmp or Fgf signaling pathway or the activation of Wnt signaling pathway, besides the reported coregulatory effects of Notch and Wnt signaling. The study of the effects of systematic combinations of pathway modulators provided more insight into hair cell regeneration from mitosis.

Extraction, characterization and antioxidant activity evaluation of polysaccharides from Smilacina japonica.

Three polysaccharides of uniform molecular weight designated as SJP1-1, SJP2-1, and SJP3-1 from Smilacina japonica were studied in this paper. Respectively, their specific optical rotations were -45°, -75°, and -35°, their polysaccharide contents were 89.22%, 93.05%, and 59.83%, their relative average molecular weights were 1.87 × 105 Da, 1.04 × 105 Da, and 1.36 × 104 Da. Three polysaccharides were all composed mainly of the three monosaccharides mannose, glucose, and galactose, which together in SJP1-1, SJP2-1, and SJP3-1 respectively made up 96.34%, 98.23%, and 91.67% of the total polysaccharide. The polysaccharide structure was maintained mostly by ß-glucose (1 → 3), ß-glucose (1 → 4), α-mannose (1 → 3), and α-galactose (1 → 4) glycosidic linkages. Scanning electron microscopy showed that SJP1-1, SJP2-1, and SJP3-1 were smooth uniform microspheres with diameters of 10-20 µm that combined to form both uniformly dispersed particles and blocky structures. Finally, antioxidant assay showed that these polysaccharides all had a strong ability to scavenge ·OH and DPPH· radicals, with the effects of purified polysaccharides being greater than those of crude polysaccharides. This first report on the properties of these polysaccharides provides a basis for further studies to shed light on the medical properties of Smilacina japonica.

Transcriptomic analysis of mouse cochleae suffering from gentamicin damage reveals the signalling pathways involved in hair cell regeneration.

There is a strong capacity for hair cell regeneration after damage in the inner ear of non-mammals. However, mammalian hair cells are substantially unable to regenerate. To obtain insights into the mechanism of this difference, we analyzed the transcriptomic changes in the mouse cochleae suffered from gentamicin damage and compared them with those in the chick cochleae suffered from the same damage. The results indicated that 2,230 genes had significantly differential expression between the gentamicin- and saline-treated mouse cochleae. Some of the differentially expressed genes were grouped into 265 signaling pathways, including the Notch, Wnt (Wingless and INT-1), Bmp (bone morphogenetic protein), FGF (fibroblast growth factor) and Shh (sonic hedgehog) pathways. Using pharmacological inhibitors or agonists of these pathways, the effects of these pathways on hair cell regeneration were further studied. The results indicated that Bmp alone and its coregulation with the Notch or Wnt signaling pathways increased the numbers of generated cells from transdifferentiation or proliferation in the mouse cochlea after damage, in addition to the reported coregulation of Notch and Wnt. Thus, this work indicates a new signaling pathway (Bmp) and its synergetic coregulation in mammalian hair cell regeneration, providing potential therapeutic targets to increase mammalian hair cell regeneration.

Responses of foliar phosphorus fractions to soil age are diverse along a 2 Myr dune chronosequence.

Plants respond to soil phosphorus (P) availability by adjusting leaf P among inorganic P (Pi) and organic P fractions (nucleic acids, phospholipids, small metabolites and a residual fraction). We tested whether phylogenetically divergent plants in a biodiversity hotspot similarly adjust leaf P allocation in response to P limitation by sampling along a 2 Myr chronosequence in southwestern Australia where nitrogen (N) limitation transitions to P limitation with increasing soil age. Total P and N, and P allocated to five chemical fractions were determined for photosynthetic organs from Melaleuca systena (Myrtaceae), Acacia rostellifera (Fabaceae) and Hakea prostrata (Proteaceae). Soil characteristics were also determined. Acacia rostellifera maintained phyllode total P and N concentrations at c. 0.5 and 16 mg g-1 DW, respectively, with a constant P-allocation pattern along the chronosequence. H. prostrata leaves allocated less P to Pi, phospholipids and nucleic acids with increasing soil age, while leaf N concentration was constant. M. systena had the greatest variation in allocating leaf P, whereas leaf N concentration decreased 20% along the chronosequence. Variation in P-allocation patterns was only partially conserved among species along the chronosequence. Such variation could have an impact on species distribution and contribute to species richness in P-limited environments.

Study of the Mechanisms by Which Aminoglycoside Damage Is Prevented in Chick Embryonic Hair Cells.

A major side effect of aminoglycoside antibiotics is mammalian hair cell death. It is thus intriguing that embryonic chick hair cells treated with aminoglycosides at embryonic day (E) 12 are insensitive to ototoxicity. To exclude some unknown factors in vivo that might be involved in preventing aminoglycoside damage to embryonic hair cells, we first cultured chick embryonic basilar papilla (BP) with an aminoglycoside antibiotic in vitro. The results indicated that the hair cells were almost intact at E12 and E14 and were only moderately damaged in most parts of the BP at E16 and E18. Generally, hair cells residing in the approximate and abneural regions were more susceptible to streptomycin damage. After incubation with gentamicin-conjugated Texas Red (GTTR), which is typically used to trace the entry route of aminoglycosides, GTTR fluorescence was not remarkable in hair cells at E12, was weak at E14, but was relatively strong in the proximal part of BP at E18. This result indicates that the amounts of GTTR that entered the hair cells are related to the degrees of aminoglycoside damage. The study further showed that the fluorescence intensity of GTTR decreased to a low level at E14 to E18 after disruption of mechanotransduction machinery, suggesting that the aminoglycoside entry into hair cells was mainly through mechanotransduction channels. In addition, most of the entered GTTR was not found to be colocalized with mitochondria even at E18. This finding provides another reason to explain why embryonic chick hair cells are insensitive to aminoglycoside damage.

The Effects of Environmental Factors on Ginsenoside Biosynthetic Enzyme Gene Expression and Saponin Abundance.

Panax ginseng C.A. Meyer is one of the most important medicinal plants in Northeast China, and ginsenosides are the main active ingredients found in medicinal ginseng. The biosynthesis of ginsenosides is regulated by environmental factors and the expression of key enzyme genes. Therefore, in this experiment, ginseng in the leaf opened stage, the green fruit stage, the red fruit stage, and the root growth stage was used as the test material, and nine individual ginsenosides and total saponins (the sum of the individual saponins) were detected by HPLC (High Performance Liquid Chromatography). There was a trend of synergistic increase and decrease, and saponin accumulation and transfer in different tissues. The expression of key enzyme genes in nine synthetic pathways was detected by real-time PCR, and the correlation between saponin content, gene expression, and ecological factors was analyzed. Correlation analysis showed that in root tissue, PAR (Photosynthetically Active Radiation) and soil water potential had a greater impact on ginsenoside accumulation, while in leaf tissue, temperature and relative humidity had a greater impact on ginsenoside accumulation. The results provide a theoretical basis for elucidating the relationship between ecological factors and genetic factors and their impact on the quality of medicinal materials. The results also have guiding significance for realizing the quality of medicinal materials.

Transcriptomic analysis of chicken cochleae after gentamicin damage and the involvement of four signaling pathways (Notch, FGF, Wnt and BMP) in hair cell regeneration.

Unlike mammalian hair cells, which are essentially unable to regenerate after damage, avian hair cells have a robust capacity for regeneration. The prerequisite for understanding the above difference is knowing the genetic programming of avian hair cell regeneration. Although the major processes have been known, the precise molecular signaling that induces regeneration remains unclear. To address this issue, we performed a high-throughput transcriptomic analysis of gene expression during hair cell regeneration in the chick cochlea after antibiotic injury in vivo. A total of 16,588 genes were found to be expressed in the cochlea, of which about 1000 genes were differentially expressed among the four groups studied, i.e., 2 days (d) or 3 d post-treatment with gentamicin or physiological saline. The differentially expressed genes were distributed across approximately one hundred signaling pathways, including the Notch, MAPK (FGF), Wnt and TGF-ß (BMP) pathways that have been shown to play important roles in embryonic development. Some differentially expressed genes (2-3 in each pathway) were further verified by qRT-PCR. After blocking Notch, FGF or BMP signaling, the number of regenerating hair cells and mitotic supporting cells increased. However, the opposite effect was observed after suppressing the Wnt pathway or enhancing BMP signaling. To our knowledge, the present study provided a relatively complete dataset of candidate genes and signaling pathways most likely involved in hair cell regeneration and should be a useful start in deciphering the genetic circuitry for inducing hair cell regeneration in the chick cochlea.

Erbin and ErbB2 play roles in the sexual differentiation of the song system nucleus HVC in bengalese finches (Lonchura Striata var. domestica).

Song control nuclei have distinct sexual differences in songbirds. However, the mechanism that underlies the sexual differentiation of song nuclei is still not well understood. Using a combination of anatomical, pharmacological, genetic, and behavioral approaches, the present study investigated the role of erbb2 (a homolog of the avian erythroblastic leukemia viral oncogene homolog 2) and the erbb2-interacting gene, erbin, in the sexual differentiation of the song nucleus HVC in the Bengalese finch. We first found that both erbin and erbb2 were expressed in the developing HVC at posthatch day (PHD) 15 in a male-biased fashion using qRT-PCR and in situ hybridization. Following the addition of a pharmaceutical inhibitor of the ErbB2 signaling pathway to the culture medium, cell proliferation in the cultured ventricle zone (VZ) that overlies the developing HVC decreased significantly. After the injection of erbin- or erbb2-interfering lentiviruses into the HVC and its overlying VZ at PHD 15, the cell proliferation in the VZ at PHD 24, the number of the differentiated neurons (Hu+ /BrdU+ or NeuN+ /BrdU+ ) in the HVC at PHD 31 or PHD 130, and the number of RA-projecting cells at PHD 130 all decreased significantly. Additionally, the adult songs displayed serious abnormalities. Finally, 173 male-biased genes were expressed in the developing HVC at PHD 15 using cDNA microarrays, of which 27.2% were Z-linked genes and approximately 20 genes were involved in the Erbin- or ErbB2-related signaling pathways. Our results provide some specific genetic factors that contribute to neurogenesis and sex differentiation in a song nucleus of songbirds. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 15-38, 2018.

Hair cell regeneration or the expression of related factors that regulate the fate specification of supporting cells in the cochlear ducts of embryonic and posthatch chickens.

Hair cells in posthatch chickens regenerate spontaneously through mitosis or the transdifferentiation of supporting cells in response to antibiotic injury. However, how embryonic chicken cochleae respond to antibiotic treatment remains unknown. This study is the first to indicate that unlike hair cells in posthatch chickens, the auditory epithelium was free from antibiotic injury (25-250 mg gentamicin/kg) in embryonic chickens, although FITC-conjugated gentamicin actually reached embryonic hair cells. Next, we examined and counted the cells and performed labeling for BrdU, Sox2, Atoh1/Math1, PV or p27(kip1) (triple or double labeling) in the injured cochlea ducts after gentamicin treatment at 2 h (h), 15 h, 24 h, 2 days (d), 3 d and 7 d after BrdU treatment in posthatch chickens. Our results indicated that following gentamicin administration, proliferating cells (BrdU+) were labeled for Atoh1/Math1 in the damaged areas 3d after gentamicin administration, whereas hair cells (PV+) renewed through mitosis (BrdU+) or direct transdifferentiation (BrdU-) were evident only after 5 d of gentamicin administration. In addition, Sox2 expression was up-regulated in triggered supporting cells at an early stage of regeneration, but stopped at the advent of mature hair cells. Our study also indicated that p27(kip1) was expressed in both hair cells and supporting cells but was down-regulated in a subgroup of the supporting cells that gave rise to hair cells. These data and the obtained dynamic changes of the cells labeled for BrdU, Sox2, Atoh1/Math1, PV or p27(kip1) are useful for understanding supporting cell behaviors and their fate specification during hair cell regeneration.