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.

Comparison of genes involved in brain development: insights into the organization and evolution of the telencephalic pallium.

The mechanisms underlying the organization and evolution of the telencephalic pallium are not yet clear.. To address this issue, we first performed comparative analysis of genes critical for the development of the pallium (Emx1/2 and Pax6) and subpallium (Dlx2 and Nkx1/2) among 500 vertebrate species. We found that these genes have no obvious variations in chromosomal duplication/loss, gene locus synteny or Darwinian selection. However, there is an additional fragment of approximately 20 amino acids in mammalian Emx1 and a poly-(Ala)6-7 in Emx2. Lentiviruses expressing mouse or chick Emx2 (m-Emx2 or c-Emx2 Lv) were injected into the ventricle of the chick telencephalon at embryonic Day 3 (E3), and the embryos were allowed to develop to E12-14 or to posthatchling. After transfection with m-Emx2 Lv, the cells expressing Reelin, Vimentin or GABA increased, and neurogenesis of calbindin cells changed towards the mammalian inside-out pattern in the dorsal pallium and mesopallium. In addition, a behavior test for posthatched chicks indicated that the passive avoidance ratio increased significantly. The study suggests that the acquisition of an additional fragment in mammalian Emx2 is associated with the organization and evolution of the mammalian pallium.

Living high training low induces physiological cardiac hypertrophy accompanied by down-regulation and redistribution of the renin-angiotensin system.

AIM: Living high training low" (LHTL) is an exercise-training protocol that refers living in hypoxia stress and training at normal level of O2. In this study, we investigated whether LHTL caused physiological heart hypertrophy accompanied by changes of biomarkers in renin-angiotensin system in rats. METHODS: Adult male SD rats were randomly assigned into 4 groups, and trained on living low-sedentary (LLS, control), living low-training low (LLTL), living high-sedentary (LHS) and living high-training low (LHTL) protocols, respectively, for 4 weeks. Hematological parameters, hemodynamic measurement, heart hypertrophy and plasma angiotensin II (Ang II) level of the rats were measured. The gene and protein expression of angiotensin-converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II receptor I (AT1) in heart tissue was assessed using RT-PCR and immunohistochemistry, respectively. RESULTS: LLTL, LHS and LHTL significantly improved cardiac function, increased hemoglobin concentration and RBC. At the molecular level, LLTL, LHS and LHTL significantly decreased the expression of ACE, AGT and AT1 genes, but increased the expression of ACE and AT1 proteins in heart tissue. Moreover, ACE and AT1 protein expression was significantly increased in the endocardium, but unchanged in the epicardium. CONCLUSION: LHTL training protocol suppresses ACE, AGT and AT1 gene expression in heart tissue, but increases ACE and AT1 protein expression specifically in the endocardium, suggesting that the physiological heart hypertrophy induced by LHTL is regulated by region-specific expression of renin-angiotensin system components.

The effects of Wnt, BMP, and Notch signaling pathways on cell proliferation and neural differentiation in a song control nucleus (HVC) of Lonchura striata.

There is obvious sexual dimorphism in the song control system of songbirds. In the higher vocal center (HVC), cell proliferation and neuronal differentiation contribute to the net addition of neurons. However, the mechanism underlying these changes is unclear. Given that Wnt, Bmp, and Notch pathways are involved in cell proliferation and neuronal differentiation, no reports are available to study the role of the three pathways in the song control system. To address the issue, we studied cell proliferation in the ventricle zone overlying the developing HVC and neural differentiation within the HVC of Bengalese finches (Lonchura striata) at posthatching day 15 when HVC progenitor cells are generated on a large scale and differentiate into neurons, after Wnt and Bmp pathways were activated by using a pharmacological agonist (LiCl) or Bmp4, respectively, and the Notch pathway was inhibited by an inhibitor (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester), DAPT). The results indicated that both cell proliferation and neural differentiation toward HVC neurons increased significantly after activation of the Wnt signaling pathway or inhibition of the Notch signaling pathway. Although cell proliferation was increased, neural differentiation was inhibited after treatment with Bmp4. There was obvious synergetic enhancement in the number of proliferating cells after the coregulation of two or three signaling pathways. In addition, synergetic enhancement was also found in the Wnt and Notch pathways in neural differentiation toward neurons within HVC. These results suggest that the three signaling pathways are involved in cell proliferation and neural differentiation of HVC.

Notch1 is involved in cell proliferation and neuronal differentiation in the HVC of zebra finch (Taeniopygia guttata).

Significant sex differences are found in songbirds' song control nuclei and their controlled song behaviors. To elucidate the underlying mechanisms, we explored the role of Notch1 during the development of the high vocal centre (HVC) and song learning in zebra finch. Our study first found that Notch1 positive cells were distributed in HVC with female-biased densities at posthatching day (PHD) 15, but male-biased at PHD 45 and adult. There were about 60 putative oestrogen-responsive elements within 2.5 kb upstream of Notch1, and Notch1 mRNA in the explants that contained the developing male HVC was significantly increased after estrogen addition into the cultured medium for 48 h. After injecting Notch1-interfering lentivirus into the male or female HVC at PHD 15, cell proliferation was significantly promoted in the ventricle zone overlying the HVC at PHD 23. In addition, neuronal differentiation towards Hu+ /BrdU+ at PHD 31, mature neurons (NeuN+/BrdU+) including those projecting to RA in HVC and the sizes of HVC and RA at adult increased significantly after Notch1-interfering lentiviruses were injected into the male HVC at PHD 15. However, the above measurements decreased, following the injection of the lentiviruses expressing Notch intracellular domain (NICD). Finally, the repeat numbers of syllables 'b' or 'c' of learned songs changed after the injection of Notch1-interfering or NICD-expressing lentiviruses into the HVC at PHD15. Our study suggests that Notch1 is related to the development of HVC and song learning in the zebra finch.

Loss of Wnt8b has no overt effect on hippocampus development but leads to altered Wnt gene expression levels in dorsomedial telencephalon.

Wnt signalling proteins regulate many aspects of animal development. We have investigated the function of mouse Wnt8b during forebrain development. Wnt8b is expressed in a highly restricted pattern including the prospective hippocampus and hypothalamus. Mutant mice lacking Wnt8b are viable and healthy. The size and morphology of the hippocampus appeared normal in mutant embryos and adults, and we found no evidence of hypothalamic defects in mutants. Wnt8b is also expressed in the neurogenic region of the adult dentate gyrus, however, cell proliferation was unchanged in Wnt8b(-/-) mutants. Mutant embryos did, however, display altered levels of expression of other Wnt genes normally expressed in forebrain. The spatial expression patterns of other Wnt genes and the overall level of canonical Wnt activity were indistinguishable from wild-types. Thus, loss of Wnt8b does not give rise to an overt morphological phenotype, but does affect expression levels of other Wnts in developing forebrain.

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.

Song control nuclei in male and female large-billed crows (Corvus macrorhynchos).

We show that the learned vocalizations of male and female large-billed crows (Corvus macrorhynchos) are similar and that their functions and physical features show significant differences from those of other oscine species. We investigate whether the song control nuclei of crows show any sexual differences in size, reflecting differences in their singing behavior, and whether these nuclei are different from those of other songbirds in terms of neural connectivity size and relative to the forebrain. Our Nissl staining results reveal that 1) of the four song nuclei examined (HVC; the robust nucleus of the arcopallium [RA]; Area X; and the dorsolateral medial nucleus [DLM]), HVC, RA, and Area X volumes are significantly larger in males than in females, but DLM volume and body and brain weights show no significant gender differences; and 2) the sizes of song nuclei relative to the forebrain are within the range of other oscines. By injecting a neural tract tracer (DiI) into various song nuclei in brain slices, we found that, as in other songbirds, HVC projects to RA and Area X, while Area X projects to the lateral magnocellular nucleus of the anterior nidopallium (IMAN) and DLM, DLM to IMAN, and IMAN to RA. Our results Indicate that, although the crow has songs very different from those of other oscine species, Its song nuclei and the connections between them are not obviously different.

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.

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.

Neurogenic development of the visual areas in the Chinese softshell turtle (Pelodiscus sinensis) and evolutionary implications.

To characterize the neurogenic development of the visual areas of the turtle (Pelodiscus sinensis) during embryogenesis, a single dose of [(3)H]-thymidine (10 microCi) was injected into egg yolks from stages S11-12 to S21. At hatching, localization of [(3)H]-thymidine incorporation was examined, and led to three main observations. (1) Neurogenesis occurred in the stratum griseum centrale of the tectum opticum from S11-12 to S16 with a peak at S12. No obvious gradients of neurogenesis were observed. (2) Neurogenesis in the nucleus rotundus (Rot) and in the dorsal lateral geniculate nucleus (GLd) occurred from S11-12 to S15. Gradients of neurogenesis were detected along ventral-dorsal and lateral-medial axes in the Rot, but only the latter neurogenic gradient occurred in the GLd. (3) In the visual region of the dorsal ventricular ridge, neurogenesis lasted from S11-12 to S16. Similarly, neurogenesis occurred from S11-12 to S16-17 in the dorsal cortex, with a peak at S12 for both telencephalic visual regions. Neurogenesis followed a ventrolateral to dorsomedial gradient in the visual region of the dorsal ventricular ridge, and a superficial to deep gradient in the caudal dorsal cortex. A significant number of neurons in the rostral dorsal cortex followed a deep (earlier arising) to superficial (later arising) pattern of neurogenesis, similar to that in the avian Wulst or in the mammalian isocortex. Finally, we compared the timing and development of neurogenesis in the turtle with birds and mammals to understand the evolutionary implications of these processes.

Comparative analysis of neurogenesis between the core and shell regions of auditory areas in the chick (Gallus gallus domesticus).

Early embryogenesis can reflect constituting organizations and evolutionary origins of brain areas. To determine whether a clear core-versus-shell distinction of neurogenesis that occurs from the auditory midbrain to the telencephalon in the reptile also appears in the bird, a single dose of [(3)H]-thymidine was injected into chick (Gallus gallus domesticus) eggs at some successive embryonic days (E) (from E3 to E10). Towards the end of hatching, [(3)H]-thymidine labeling was examined, and the results were as follows: 1) Neuronal generation in the nucleus intercollicularis (ICo) (shell region) began at E3, whereas neurogenesis began at E4 in the nucleus mesencephalicus lateralis pars dorsalis (MLd) (core region); 2) Neurogenesis initiated at E3 in the nucleus ovoidalis (Ov) shell, but initiated at E4 in the rostral Ov core. In the medial or caudal Ov core, the percentage of heavily-labeled neurons with [(3)H]-thymidine was significantly lower at E3 age group than that in the Ov shell; 3) In field L1 and L3, two flanking regions of the primary telencephalic auditory area (field L2a), neurogenesis started at E5, but started at E6 in field L2a. These data indicate that the onset of embryogenesis began earlier in the auditory shell areas than in the core areas from the midbrain to the telencephalon. These findings provide insight into the organization of auditory nuclei and their evolution in amniotes.

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.

Comparative analyses of song complexity and song-control nuclei in fourteen oscine species.

Most studies on the relationship between measures of song behavior and the sizes of song control nuclei have focused on one or two oscine species, and often show inconsistent results. To address this issue, we first measured four variables for song complexity, i.e., song repertoire size, syllable repertoire size, the mean number of syllables per phrase (MNS) and the number of syllables in the longest phrase (NSLP), and the sizes of three song control nuclei, i.e., HVC, RA (the robust nucleus of the arcopallium), and Area X in 14 oscine species from eight families. To tackle the problem of statistical non-independence that probably existed among the closely related species, we reconstructed the phylogeny of the species studied using mitochondrial cytochrome b DNA sequences from GenBank. By using the methods adopted in most previous reports, we tested the relationship between song complexity and the sizes of the song control nuclei. We found that: 1) the absolute sizes of RA and Area X, but not of HVC, were positively correlated to the three measures of song complexity, but that only the residual size of RA in regard to telencephalon size was significantly correlated to the song measures; 2) independent contrasts analysis showed RA and Area X to be significantly associated with NSLP. Our results indicated that the relationship between song behavior and its neural structures varied among song nuclei, suggesting that each song control nucleus may play a different role in song behavior.

Distinction of neurochemistry between the cores and their shells of auditory nuclei in tetrapod species.

The distribution of Met-enkephalin (ENK), substance P (SP) and serotonin (5-HT) differs between the core and shell regions of the mesencephalic and diencephalic auditory nuclei of the turtle [Belekhova et al., 2002]. These neurochemical distinctions are also found in other tetrapods (mammals, birds and amphibians). The distribution of ENK, SP and 5-HT was examined in the core and shell regions of both mesencephalic and diencephalic auditory nuclei, and in the telencephalic auditory areas of Bengalese finches (Lonchura striata) and mice (Mus musculus), as well as in corresponding auditory areas in toads (Bufo bufo). ENK, SP and 5-HT immunoreactive fibers and perikarya were largely absent from the core regions of both mesencephalic and diencephalic auditory nuclei, in comparison with the shell regions of mice and Bengalese finches. In the toad, however, this pattern was observed in the mesencephalic auditory nucleus, but not in the diencephalic auditory areas. ENK and SP immunoreactive perikarya were detected in the telencephalic auditory area of mice, whereas no ENK, SP or 5-HT immunolabeling was observed in the telencephalic auditory area (Field L) of Bengalese finches. These findings are discussed in terms of the evolution of the core-and-shell organization of auditory nuclei of tetrapods.

Differences in neurogenesis differentiate between core and shell regions of auditory nuclei in the turtle (Pelodiscus sinensis): evolutionary implications.

There is a clear core-versus-shell distinction in cytoarchitecture, electrophysiological properties and neural connections in the mesencephalic and diencephalic auditory nuclei of amniotes. Determining whether the embryogenesis of auditory nuclei shows a similar organization is helpful for further understanding the constituent organization and evolution of auditory nuclei. Therefore in the present study, we injected [(3)H]-thymidine into turtle embryos (Pelodiscus sinensis) at various stages of development. Upon hatching, [(3)H]-thymidine labeling was examined in both the core and shell auditory regions in the midbrain, diencephalon and dorsal ventricular ridge. Met-enkephalin and substance P immunohistochemistry was used to distinguish the core and shell regions. In the mesencephalic auditory nucleus, the occurrence of heavily labeled neurons in the nucleus centralis of the torus semicircularis reached its peak at embryonic day 9, one day later than the surrounding shell. In the diencephalic auditory nucleus, the production of heavily labeled neurons in the central region of the reuniens (Re) was highest at embryonic day (E) 8, one day later than that in the shell region of reuniens. In the region of the dorsal ventricular ridge that received inputs from the central region of Re, the appearance of heavily labeled neurons also reached a peak one day later than that in the area receiving inputs from the shell region of reuniens. Thus, there is a core-versus-shell organization of neuronal generation in reptilian auditory areas.

Dynamic changes of apoptosis and expression of Bcl-2 family members in the posthatch hippocampus of Bengalese finches.

The hippocampus of songbirds plays an important role in spatial memory, and probably in song learning. Although prolonged neuronal generation and apoptosis are thought to be closely correlated with memory function, natural changes of the number of neurons and in apoptosis in the hippocampus of songbirds have not been fully investigated during development and in the adult. In the current study, we examined developmental changes in the volume and the number of neurons and apoptotic cells in the hippocampus of songbirds (Lonchura striata) from posthatch day (P5) to adulthood. Apoptotic cells were determined by Nissl staining and immunohistochemistry for cleaved caspase-3, a key apoptotic caspase executioner. The expression levels of Bcl-2 family member mRNA and protein, including Bcl-2, Bcl-xL and Bax, were also investigated. Our results indicated that: (1) the hippocampus volume significantly increased from P5 to P60, although the number of neurons remained stable in all studied stages; (2) the number of apoptotic cells was highest at P45, based either on the Nissl staining or on the immunohistochemistry for caspase-3; (3) Bcl-2 mRNA expression was high from P5 to adulthood, while Bax mRNA declined abruptly from P5 to adulthood, and Bcl-x mRNA was high after P45. Bcl-2 protein was only detected at P5 and P15, while detection of Bcl-xL and Bax proteins paralleled levels of mRNA expression. Our study provides detailed changes of apoptosis in the posthatch songbird hippocampus, suggesting an important role for caspase-3 and Bcl-2 family members in hippocampus apoptosis.

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.

Sexual Differences in Cell Loss during the Post-Hatch Development of Song Control Nuclei in the Bengalese Finch.

Birdsongs and the regions of their brain that control song exhibit obvious sexual differences. However, the mechanisms underlying these sexual dimorphisms remain unknown. To address this issue, we first examined apoptotic cells labeled with caspase-3 or TUNEL in Bengalese finch song control nuclei - the robust nucleus of the archopallium (RA), the lateral magnocellular nucleus of the anterior nidopallium (LMAN), the high vocal center (HVC) and Area X from post-hatch day (P) 15 to 120. Next, we investigated the expression dynamics of pro-apoptotic (Bid, Bad and Bax) and anti-apoptotic (Bcl-2 and Bcl-xL) genes in the aforementioned nuclei. Our results revealed that the female RA at P45 exhibited marked cell apoptosis, confirmed by low densities of Bcl-xL and Bcl-2. Both the male and female LMAN exhibited apoptotic peaks at P35 and P45, respectively, and the observed cell loss was more extensive in males. A corresponding sharp decrease in the density of Bcl-2 after P35 was observed in both sexes, and a greater density of Bid was noted at P45 in males. In addition, we observed that RA volume and the total number of BDNF-expressing cells decreased significantly after unilateral lesion of the LMAN or HVC (two areas that innervate the RA) and that greater numbers of RA-projecting cells were immunoreactive for BDNF in the LMAN than in the HVC. We reasoned that a decrease in the amount of BDNF transported via HVC afferent fibers might result in an increase in cell apoptosis in the female RA. Our data indicate that cell apoptosis resulting from different pro- and anti-apoptotic agents is involved in generating the differences between male and female song control nuclei.