Sexual Differences in Cell Proliferation in the Ventricular Zone,Cell Migration and Differentiation in the HVC of Juvenile Bengalese Finch

Song control nuclei have distinct sexual differences and thus are an ideal model to address how brain areas are sexually differentiated. Through a combination of histological analysis and electrical lesions, we first identified the ventricle site for HVC progenitor cells. We then found that there were significant sex differences in the cellular proliferation activity in the ventricular zone of the HVC, the number of migrating cells along the radial cells (positive immunoreactions to vimentin) and differentiation towards neurons. Through co-culturing of male and female slices containing the developing HVC in the same well, we found that the male slices could produce diffusible substances to masculinize the female HVC. By adding estrogen, an estrogen antagonist, brain-derived neurotrophic factor (BDNF) or its antibody into the culture medium, separately or in combination, we found that these diffusible substances may include estrogen and BDNF. Finally, we found that 1) estrogen-induced BDNF upregulation could be detected 48 hr after estrogen treatment and could not be blocked by a vascular endothelial growth factor (VEGF) receptor inhibitor and 2) the amount of VEGF mRNA expressed in the developing HVC and its adjacent area did not display any significant sex differences, as did the distribution of VEGF and laminin-expressing endothelial cells in the developing HVC. Because these findings are largely different from previous reports on the adult female HVC, it is suggested that our estrogen-induced BDNF up-regulation and the resultant sexual differentiation might not be mediated by VEGF and endothelial cells, but instead, may result from the direct effects of estrogen on BDNF.     

Early Rearing Conditions Affect the Development of Body Size and Song in Bengalese Finches

Birdsong is an acoustic ornament. According to indicator models, a trait must be costly to act as an honest signal, but the potential costs of elaborate songs are still poorly understood. The developmental stress hypothesis suggests that learned song characteristics could be an honest indicator of early developmental conditions because the brain structures associated with learning songs are susceptible to early developmental stress, which could thus affect song development. Unlike previous studies of developmental stress that examined the effect of a stress hormone or restricted nutrition, we observed Bengalese finches under semi‐natural breeding conditions in captivity to investigate the relationship between early rearing conditions (e.g., brood size and sex ratio) and the subsequent variation in body size and song among individuals. Our results suggest that the early rearing environment directly affects body size and song complexity, whereas song output is determined mainly by body size. These results support the developmental stress hypothesis. Moreover, our findings are the first to show that developmental condition affects not only the number of note types but also the syntactical complexity of the song.     

10种鸣禽鸣唱复杂性与发声核团体积的聚类分析

选用捕自野外和人工繁殖的10种雄性成鸟(一年龄以上)作为实验材料。当鸟适应环境后录音,用VS-99语音工作站软件进行声谱分析。鸣唱的复杂性采用语句短语总数、短语的音节数之和、短语的音节种类数之和、每个短语中所含的平均音节数、每个短语中所含的平均音节种类数、每种鸣禽最长短语的音节数和最长短语的音节种类数7项指标表示。然后测定前脑的上纹状体腹侧尾端(HVC)、古纹状体粗核(RA)以及嗅叶的X核(Areax)3个主要鸣唱控制核团的体积。最后分别对10种鸣禽3个发声控制核团体积和鸣唱复杂性的7项指标进行聚类分析。10种鸣禽的7项指标值相差较大,即使同一科也如此。蒙古百灵的3种核团体积比值均最大,其次是金丝雀和黄喉鹉。10种鸣禽鸣唱语句复杂性的7个指标和3种核团体积聚类分析树形图显示的结果各不相同;仅RA和Areax核团体积的树形图显示蒙古百灵远离其他9种鸣禽,与现代分类学和DNA分析得到的进化树一致。     

Learning-Related Neuronal Activation in the Zebra Finch Song System Nucleus HVC in Response to the Bird's Own Song

Like many other songbird species, male zebra finches learn their song from a tutor early in life. Song learning in birds has strong parallels with speech acquisition in human infants at both the behavioral and neural levels. Forebrain nuclei in the 'song system' are important for the sensorimotor acquisition and production of song, while caudomedial pallial brain regions outside the song system are thought to contain the neural substrate of tutor song memory. Here, we exposed three groups of adult zebra finch males to either tutor song, to their own song, or to novel conspecific song. Expression of the immediate early gene protein product Zenk was measured in the song system nuclei HVC, robust nucleus of the arcopallium (RA) and Area X. There were no significant differences in overall Zenk expression between the three groups. However, Zenk expression in the HVC was significantly positively correlated with the strength of song learning only in the group that was exposed to the bird's own song, not in the other two groups. These results suggest that the song system nucleus HVC may contain a neural representation of a memory of the bird's own song. Such a representation may be formed during juvenile song learning and guide the bird's vocal output.     

Undirected Song Encourages the Breeding Female Zebra Finch to Remain in the Nest

Wild zebra finches sing frequently during the breeding season, but the vast majority of song is of the undirected song type that is not directed at any individual, and the function of which is obscure — it appears to be ignored by all potential recipients. It is sung close to the nest-site, has a peak in production during the egg-laying period, and diminishes thereafter. The incidence of undirected song is positively correlated with extra-pair courtship, a finding consistent with the hypothesis that it is a means of advertising availability for extra-pair matings. Typically, undirected song occurred outside the nest when the female was inside, and a positive relationship was found between the amount of singing given by the male during the 5-min interval immediately after the female entered the nest and the time she subsequently spent inside the nest. Keeping the partner inside the nest during her fertile period is an advantage to the male: it serves as a form of paternity protection against other males and it allows him opportunities to pursue his own extra-pair matings. Occupancy of the nest during laying is also a means of guarding against intraspecific brood parasitism, which was high at this colony.     

The Problem of Nucleolar Number in Cell Nuclei and in Sections of Cell Nuclei

In this paper the mean number N of nucleoli of cell nuclei and the relative frequency PN of cell nuclei with N nucleoli are investigated. To solve the first problem, the stereological model of convex shaped nuclei and nucleoli and the assumption of randomly-isotropically distributed objects in space are used. The model is developed for non-vanishing thickness T of tissue sections. The relationship between the unknown number N of nucleoli in cell nuclei and the mean number n of nucleoli in nuclear sections determined by counting is N = n (D+T)/(d + T). Here D and d are the mean values of caliper diameter for nuclei and for nucleoli, resp. The second problem is illustrated by means of some biomedical examples: The relative frequency PN of cell nuclei with N nucleoli can be approximated by a generalized Poisson distribution in all investigated cases. Therefore the mean nucleolar number N is the essential parameter to describe the frequencies of cell nuclei with different numbers of nucleoli.     

白腰文鸟发声相关核团体积在发育中的变化及神经机制

首先研究了5-120日龄雌雄白腰文鸟（Lonchura striata swinhoei)4个主要发声核团（RA,LMAN,AreaX和HVC）的体积变化,再通过神经示踪技术研究这些核团与其他核团神经联系的建立时间,以了解发声核团发育及性别分化的神经机制,结果表明：（1）雌雄RA体积均在20,30日龄前后表现出急剧的变化和雌雄差异;雌雄RA在15和25日龄分别接受LMAN和HVC的神经支配,（2）雌雄LMAN体积分别在20,30日龄前先增长,之后均缩小,雌雄LMAN的神经元大小均在15和20日龄间急剧增长,但在该时段之后,不再发生明显变化,雌雄LMAN均在15日龄接受RA的神经支配。（3）AreaX核体积,神经元大小最明显的变化位于20-25日龄间;雌雄AreaX核均在15日龄时接受HVC的神经支配,（4）AreaX核体积,神经元大小最明显的变化位于20-25日龄间;雌雄AreaX核均在15日龄时接受HVC的神经支配,（4）雌雄HVC体积变化的最大值在20和30日龄前后,雄乌HVC的神经元大小在20,30日龄前后,雌鸟在15-20日龄发生较大的变化,其余组间变化小或不明显,雌雄HVC分别在15,25日龄同AreaX核,RA建立神经联系,因此,4个发声核团组织学的明显变化与核团间神经联系的建立相关,说明发声核团间的神经联系可能影响和决定了核团体积在发育中的变化。     

Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

Activation-induced deaminase (AID) converts DNA cytosines to uracils in immunoglobulin genes, creating antibody diversification. It also causes mutations and translocations that promote cancer. We examined the interplay between uracil creation by AID and its removal by UNG2 glycosylase in splenocytes undergoing maturation and in B cell cancers. The genomic uracil levels remain unchanged in normal stimulated B cells, demonstrating a balance between uracil generation and removal. In stimulated UNG^−/− cells, uracil levels increase by 11- to 60-fold during the first 3 days. In wild-type B cells, UNG2 gene expression and enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is coordinated with uracil creation by AID. Remarkably, a murine lymphoma cell line, several human B cell cancer lines, and human B cell tumors expressing AID at high levels have genomic uracils comparable to those seen with stimulated UNG^−/−splenocytes. However, cancer cells express UNG2 gene at levels similar to or higher than those seen with peripheral B cells and have nuclear uracil excision activity comparable to that seen with stimulated wild-type B cells. We propose that more uracils are created during B cell cancer development than are removed from the genome but that the uracil creation/excision balance is restored during establishment of cell lines, fixing the genomic uracil load at high levels.     

红豆草根瘤侵染细胞核在细胞凋亡中的超微结构变化

用透射电镜观察红豆草根瘤侵染细胞核在细胞凋亡过程中的超微结构,以探讨红豆草根瘤侵染细胞核在发育过程中的超微结构变化及其与细胞凋亡的关系.结果表明,红豆草根瘤侵染细胞核的超微结构随细胞发育程度不同而不同.在幼龄侵染细胞中,细胞核体积较大,近似圆形.在即将成熟和成熟的侵染细胞中,细胞核膜有内陷现象,其核仁常具有核仁泡和核仁联合体.在早期凋亡的侵染细胞中,细胞核体积减小,形状变得不规则,核膜出现大量内陷,在其表面形成许多大的突起和深的沟槽,有时还有内质网、线粒体、小液泡和细菌等位于核膜的内陷处,而且核仁也开始裂解.在后期凋亡的侵染细胞中,除细菌解体外,还出现核仁消失,核膜破裂,核质外流,并在细胞质中形成一些电子密度很高,无一定形状的团块状物质.     

Cell Differentiation during Sexual Development of the Fungus Sordaria macrospora Requires ATP Citrate Lyase Activity

During sexual development, mycelial cells from most filamentous fungi differentiate into typical fruiting bodies. Here, we describe the isolation and characterization of the Sordaria macrospora developmental mutant per5, which exhibits a sterile phenotype with defects in fruiting body maturation. Cytological investigations revealed that the mutant strain forms only ascus precursors without any mature spores. Using an indexed cosmid library, we were able to complement the mutant to fertility by DNA-mediated transformation. A single cosmid clone, carrying a 3.5-kb region able to complement the mutant phenotype, has been identified. Sequencing of the 3.5-kb region revealed an open reading frame of 2.1 kb interrupted by a 66-bp intron. The predicted polypeptide (674 amino acids) shows significant homology to eukaryotic ATP citrate lyases (ACLs), with 62 to 65% amino acid identity, and the gene was named acl1. The molecular mass of the S. macrospora ACL1 polypeptide is 73 kDa, as was verified by Western blot analysis with a hemagglutinin (HA) epitope-tagged ACL1 polypeptide. Immunological in situ detection of the HA-tagged polypeptide demonstrated that ACL is located within the cytosol. Sequencing of the mutant acl1 gene revealed a 1-nucleotide transition within the coding region, resulting in an amino acid substitution within the predicted polypeptide. Further evidence that ACL1 is essential for fruiting body maturation comes from experiments in which truncated and mutated versions of the acl1 gene were used for transformation. None of these copies was able to reconstitute the fertile phenotype in transformed per5 recipient strains. ACLs are usually involved in the formation of cytosolic acetyl coenzyme A (acetyl-CoA), which is used for the biosynthesis of fatty acids and sterols. Protein extracts from the mutant strain showed a drastic reduction in enzymatic activity compared to values obtained from the wild-type strain. Investigation of the time course of ACL expression suggests that ACL is specifically induced at the beginning of the sexual cycle and produces acetyl-CoA, which most probably is a prerequisite for fruiting body formation during later stages of sexual development. We discuss the contribution of ACL activity to the life cycle of S. macrospora.     

Basic Proteins of Plant Nuclei during Normal and Pathological Cell Growth

Histone proteins were studied by microphotometry of plant tissue sections stained with fast green at pH 8.1. For comparative purposes the Feulgen reaction was used for deoxyribose nuclei acid (DNA); the Sakaguchi reaction for arginine; and the Millon reaction for estimates of total protein. Analysis of Tradescantia tissues indicated that amounts of nuclear histone fell into approximate multiples of the gametic (egg or sperm) quantity except in dividing tissues, where amounts intermediate between multiples were found. In differentiated tissues of lily, corn, onion, and broad bean, histones occurred in constant amounts per nucleus, characteristic of the species, as was found also for DNA. Unlike the condition in several animal species, the basic proteins of sperm nuclei in these higher plants were of the histone type; no evidence of protamine was found. In a plant neoplasm, crown gall of broad bean, behavior of the basic nuclear proteins closely paralleled that of DNA. Thus, alterations of DNA levels in tumor tissues were accompanied by quantitatively similar changes in histone levels to maintain the same Feulgen/fast green ratios found in homologous normal tissues.     

Sexual Dimorphism of the Zebra Finch Syrinx Indicates Adaptation for High Fundamental Frequencies in Males

Background

In many songbirds the larger vocal repertoire of males is associated with sexual dimorphism of the vocal control centers and muscles of the vocal organ, the syrinx. However, it is largely unknown how these differences are translated into different acoustic behavior.

Methodology/Principal Findings

Here we show that the sound generating structures of the syrinx, the labia and the associated cartilaginous framework, also display sexual dimorphism. One of the bronchial half rings that position and tense the labia is larger in males, and the size and shape of the labia differ between males and females. The functional consequences of these differences were explored by denervating syringeal muscles. After denervation, both sexes produced equally low fundamental frequencies, but the driving pressure generally increased and was higher in males. Denervation strongly affected the relationship between driving pressure and fundamental frequency.

Conclusions/Significance

The syringeal modifications in the male syrinx, in concert with dimorphisms in neural control and muscle mass, are most likely the foundation for the potential to generate an enhanced frequency range. Sexually dimorphic vocal behavior therefore arises from finely tuned modifications at every level of the motor cascade. This sexual dimorphism in frequency control illustrates a significant evolutionary step towards increased vocal complexity in birds.     

Undirected Song in Wild Zebra Finch Flocks: Contexts and Effects of Mate Removal

Wild zebra finches, Taeniopygia guttata, sing frequently throughout the year but the function of undirected song, the most prevalent type, is unknown. Paired males commonly sang undirected song in feeding flocks where it occurred more frequently in the non-breeding season than in the breeding season. Song rate varied greatly among males. The context of the singer and the absence of any overt responses from flock members excludes direct aggressive and mate-attracting functions. Performance of undirected song may have energetic costs and predatory risks. The finding that singers are active individuals with ‘spare time’, that are neither hungry, nor tired, nor sexually motivated, suggests that singers are advertising their ‘quality’ as potential mates and/or participants for extra-pair copulations. When the female partner in non-breeding pairs was experimentally removed, the rate of undirected song of the ‘widower’ increased significantly. Males quickly re-paired. This experiment supports the hypothesis that singing in the flock has a mate-attracting role.     

A Daily Oscillation in the Fundamental Frequency and Amplitude of Harmonic Syllables of Zebra Finch Song

Complex motor skills are more difficult to perform at certain points in the day (for example, shortly after waking), but the daily trajectory of motor-skill error is more difficult to predict. By undertaking a quantitative analysis of the fundamental frequency (FF) and amplitude of hundreds of zebra finch syllables per animal per day, we find that zebra finch song follows a previously undescribed daily oscillation. The FF and amplitude of harmonic syllables rises across the morning, reaching a peak near mid-day, and then falls again in the late afternoon until sleep. This oscillation, although somewhat variable, is consistent across days and across animals and does not require serotonin, as animals with serotonergic lesions maintained daily oscillations. We hypothesize that this oscillation is driven by underlying physiological factors which could be shared with other taxa. Song production in zebra finches is a model system for studying complex learned behavior because of the ease of gathering comprehensive behavioral data and the tractability of the underlying neural circuitry. The daily oscillation that we describe promises to reveal new insights into how time of day affects the ability to accomplish a variety of complex learned motor skills.     

Melatonin Binding in the House Sparrow Song Control System: Sexual Dimorphism and the Effect of Photoperiod

Avian song is a sexually dimorphic behavior which is regulated seasonally. This regulation involves the construction and growth of song control structures: the high vocal center (HVC),nucleus robustus archistrialis(RA),nucleus magnocellularis anterior(MAN), and Area X. Song behavior and its neural correlates are controlled by steroid-dependent and independent processes. The avian circadian system is known to be involved in both daily processes and seasonal reproduction. A major part of this system is the circadian secretion of melatonin by the pineal gland. To determine possible interactions of the circadian and song control systems, the distribution and density of 2-[¹²⁵I]iodomelatonin (IMEL) binding, an indicator of melatonin sensitivity, were determined in male and female house sparrow brains. Specific binding was found in visual system centers of both genders, but binding in HVC, RA, and Area X was present only in males. Binding in MAN was present in both sexes. Although the effects of short and long photoperiods on male house sparrow IMEL binding in song structures revealed no systematic changes, there were significant differences in binding under different photoperiods in HVC and RA. IMEL binding in the tectofugalnucleus rotundus,however, was consistently highest under short day conditions. IMEL binding in song control nuclei was independent of testicular influence, since castration did not affect it significantly. The data point to a role for the circadian system of house sparrows in song control, but a specific role for melatonin in the daily or seasonal regulation of the song control system in birds, could not be determined.     

Loss of Cell Cycle Checkpoint Control in Drosophila Rfc4 Mutants

Two alleles of the Drosophila melanogaster Rfc4 (DmRfc4) gene, which encodes subunit 4 of the replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role in DNA replication but also result in mitotic chromosomal defects appearing either as premature chromosome condensation-like or precocious sister chromatid separation figures. Though the DmRFC4 protein localizes to all replicating nuclei, it is dispersed from chromatin in mitosis. Thus the mitotic defects appear not to be the result of a direct role for RFC4 in chromosome structure. We also show that the mitotic defects in these two DmRfc4 alleles are the result of aberrant checkpoint control in response to DNA replication inhibition or damage to chromosomes. Not all surveillance function is compromised in these mutants, as the kinetochore attachment checkpoint is operative. Intriguingly, metaphase delay is frequently observed with the more severe of the two alleles, indicating that subsequent chromosome segregation may be inhibited. This is the first demonstration that subunit 4 of RFC functions in checkpoint control in any organism, and our findings additionally emphasize the conserved nature of RFC's involvement in checkpoint control in multicellular eukaryotes.