Serotonin signaling is a very early step in patterning of the left-right axis in chick and frog embryos

BACKGROUND: Consistent left-right (LR) asymmetry is a fascinating problem in developmental and evolutionary biology. Conservation of early LR patterning steps among vertebrates as well as involvement of nonprotein small-molecule messengers are very poorly understood. Serotonin (5-HT) is a key neurotransmitter with crucial roles in physiology and cognition. We tested the hypothesis that LR patterning required prenervous serotonin signaling and characterized the 5-HT pathway in chick and frog embryos. RESULTS: A pharmacological screen implicated endogenous signaling through receptors R3 and R4 and the activity of monoamine oxidase (MAO) in the establishment of correct sidedness of asymmetric gene expression and of the viscera in Xenopus embryos. HPLC and immunohistochemistry analysis indicates that Xenopus eggs contain a maternal supply of serotonin that is progressively degraded during cleavage stages. Serotonin's dynamic localization in frog embryos requires gap junctional communication and H,K-ATPase function. Microinjection of loss- and gain-of-function constructs into the right ventral blastomere randomizes asymmetry. In chick embryos, R3 and R4 activity is upstream of the asymmetry of Sonic hedgehog expression. MAO is asymmetrically expressed in the node. CONCLUSIONS: Serotonin is present in very early chick and frog embryos. 5-HT pathway function is required for normal asymmetry and is upstream of asymmetric gene expression. The microinjection data reveal asymmetry existing in frog embryos by the 4-cell stage and suggest novel intracellular 5-HT mechanisms. These functional and localization data identify a novel role for the neurotransmitter serotonin and implicate prenervous serotonergic signaling as an obligate aspect of very early left-right patterning conserved to two vertebrate species.     

Cloning of the Retinoblastoma cDNA from the Japanese medaka (Oryzias latipes) and preliminary evidence of mutational alterations in chemically-induced retinoblastomas

We have cloned a medaka homolog of the human retinoblastoma (Rb) susceptibility gene. The medaka Rb cDNA encodes a predicted protein of 909 amino acids. DNA sequence analysis with other vertebrate Rb sequences demonstrates that the medaka Rb cDNA is highly conserved in regions of functional importance. An antibody raised against an epitope of the human pRb recognizes the protein product of the medaka Rb gene, detecting a 105 kDa protein in all tissues examined and at differential levels for the stages of embryonic development studied. The sequence reported herein, combined with the high degree of conservation observed in critical domains, has also facilitated a preliminary investigation of the molecular etiology of chemically-induced retinoblastoma. The mutational alterations characterized suggest that medaka may provide a novel model and, thus, provide additional insight into the human retinoblastoma condition.     

生长抑制素能神经元在人胎丘脑网状核内的分布

本文用免疫细胞化学方法调查了生长抑素（ＳＯＭ）免疫反应神经元在人胎丘脑网状核内的分布。流产的胚胎３例,胎龄分别为１８周,２３周,３２周。意外死亡足月新生儿１例、在１８周胚胎的丘脑网状核内可见少数染色较浅的ＳＯＭ免疫反应阳性神经元,呈圆形。从１８周到３２周,ＳＯＭ免疫反应阳性细胞数明显增多,突起更丰富。在足月新生儿,ＳＯＭ阳性细胞数较３２周有所减少。结果表明,ＳＯＭ阳性神经元存在于人胎丘脑网状核内,并且有一定的发育过程。出现于人脑发育的早期阶段,可能在中枢神经系统的发育过程中起重要的作用。     

Reception and extrareceptor binding of cytostatic serotonin antagonists by early embryos of the sea urchin Arbacia lixula

Unfertilized eggs and early embryos of the sea urchin Arbacia lixula incubated for 60 min in a medium containing the antagonists of prenervous serotonin, i.e. inmecarb (21 microM) or imipramine (40 microM), bind up to 5 microM of these drugs per 1 ml of cells. At high cell concentrations (more than 10,000 eggs or embryos per 1 ml), this binding is not followed by inhibition of cleavage divisions or by increase in the sensitivity to cytostatic effects of these drugs, which is taken as an indication that this binding is a nonreceptive one. The decrease in concentration of eggs or embryos does not affect total binding of the drugs, although their antiserotonin effects become evident indicating the existence of the receptor sites of binding. In experiments with 3H-imipramine, two binding pools were found (Bmax being correspondingly equal to about 20 and 0.75 microM/ml of embryos; the values of Kd amount to 200 and 15 microM). One of them is a nonreceptive pool, whereas the other presumably coincides with receptor binding sites of prenervous serotonin antagonists.     

人胎心肌原癌基因c-myc和jun表达的原位杂交研究

应用地高辛配体(digoxigenin)标记探针的原位杂交技术,检测了原癌基因 c-myc 和 jun在12例不同发育阶段的人胎心肌中的表达。根据胎龄,将其中9例分成三组,胎龄分别是16—17、23—24和27—28周,每组各3例。另外有32和36周及足月的大胎龄标本各例。杂交信号用图象分析仪(MIAS 300)分析处理。结果显示:c-myc mRNA 或 jun mRNA 的表达水平在3组心肌样本中的任意2组间的变化有差异显著性(P<0.01)结果表明,在胎儿早期,c-myc 和 jun 的表达都较高,随着胎龄的增加,其表达水平逐渐下降,在足月胎儿表达水平很低。结果提示,在人胎心肌的发育过程中,这两种原癌基因的表达均是一种下行调节方式,反应了 c-myc 和 jun 在心肌细胞的发育过程中与心肌细胞的生长密切相关。可能在心肌细胞的增殖与分化中起着重要调控作用。本研究也表明,地高辛配体标记探针的原位杂交技术具有灵敏度高,分辨力好和实验周期短等优点。对在体正常心肌发育的原癌基因表达的研究,有助于对正常心肌生长以及对心肌肥大发生的分子机制的了解。     

Genetic variation at the vlsE locus of Borrelia burgdorferi within ticks and mice over the course of a single transmission cycle

The Lyme disease spirochete, Borrelia burgdorferi, causes a persistent infection in the vertebrate host even though infected animals mount an active immune response against the spirochete. One strategy used by the spirochete to evade vertebrate host immunity is to vary the structure and expression of outer membrane antigens. The vlsE locus represents the best-studied example of antigenic variation in B. burgdorferi. During vertebrate host infection, recombination between the active vlsE locus and silent, partial vlsE copies leads to gene conversion events and the generation of novel alleles at the expression site. In the present study, we followed a population of B. burgdorferi organisms moving through vertebrate host and tick stages to complete one transmission cycle. The major goal of the study was to determine if the vlsE locus was subject to different selective pressure and/or recombination frequency at different stages of the spirochete's life cycle. We report here that the vlsE genetic diversity generated within the rodent host was maintained through the larval and nymphal tick stages. Therefore, naturally infected ticks are likely to transmit spirochete populations with multiple vlsE alleles into naive vertebrate hosts. Although vlsE genetic diversity in mice was maintained through tick stages, the dominant vlsE alleles were different between tick stages as well as between individual ticks. We propose that population-level bottlenecks experienced by spirochetes, especially during the larval-to-nymphal molt, are responsible for individual infected ticks harboring different dominant vlsE alleles. Although vlsE genetic diversity is maintained through tick stages, the VlsE protein is unlikely to be of functional importance in the vector, because the protein was expressed by very few (<1%) bacteria in the vector.     

Identifying a window of vulnerability during fetal development in a maternal iron restriction model

It is well acknowledged from observations in humans that iron deficiency during pregnancy can be associated with a number of developmental problems in the newborn and developing child. Due to the obvious limitations of human studies, the stage during gestation at which maternal iron deficiency causes an apparent impairment in the offspring remains elusive. In order to begin to understand the time window(s) during pregnancy that is/are especially susceptible to suboptimal iron levels, which may result in negative effects on the development of the fetus, we developed a rat model in which we were able to manipulate and monitor the dietary iron intake during specific stages of pregnancy and analyzed the developing fetuses. We established four different dietary-feeding protocols that were designed to render the fetuses iron deficient at different gestational stages. Based on a functional analysis that employed Auditory Brainstem Response measurements, we found that maternal iron restriction initiated prior to conception and during the first trimester were associated with profound changes in the developing fetus compared to iron restriction initiated later in pregnancy. We also showed that the presence of iron deficiency anemia, low body weight, and changes in core body temperature were not defining factors in the establishment of neural impairment in the rodent offspring.Our data may have significant relevance for understanding the impact of suboptimal iron levels during pregnancy not only on the mother but also on the developing fetus and hence might lead to a more informed timing of iron supplementation during pregnancy.     

FishNet: an online database of zebrafish anatomy

Background  

Over the last two decades, zebrafish have been established as a genetically versatile model system for investigating many different aspects of vertebrate developmental biology. With the credentials of zebrafish as a developmental model now well recognized, the emerging new opportunity is the wider application of zebrafish biology to aspects of human disease modelling. This rapidly increasing use of zebrafish as a model for human disease has necessarily generated interest in the anatomy of later developmental phases such as the larval, juvenile, and adult stages, during which many of the key aspects of organ morphogenesis and maturation take place. Anatomical resources and references that encompass these stages are non-existent in zebrafish and there is therefore an urgent need to understand how different organ systems and anatomical structures develop throughout the life of the fish.     

The 2R hypothesis and the human genome sequence

One theory formalised in 1970 proposes that the complexity of vertebrate genomes originated by means of genome duplication at the base of the vertebrate lineage. Since then, the theory has remained both popular and controversial. Here we review the theory, and present preliminary results from our analysis of duplications in the draft human genome sequence. We find evidence for extensive duplication of parts of the genome. We also question the validity of the 'parsimony test' that has been used in other analyses.     

Discovery of 342 putative new genes from the analysis of 5'-end-sequenced full-length-enriched cDNA human transcripts

In this work we describe the process that, starting with the production of human full-length-enriched cDNA libraries using the CAP-Trapper method, led us to the discovery of 342 putative new human genes. Twenty-three thousand full-length-enriched clones, obtained from various cell lines and tissues in different developmental stages, were 5'-end sequenced, allowing the identification of a pool of 5300 unique cDNAs. By comparing these sequences to various human and vertebrate nucleotide databases we found that about 40% of our clones extended previously annotated 5' ends, 662 clones were likely to represent splice variants of known genes, and finally 342 clones remained unknown, with no or poor functional annotation. cDNA-microarray gene expression analysis showed that 260 of 342 unknown clones are expressed in at least one cell line and/or tissue. Further analysis of their sequences and the corresponding genomic locations allowed us to conclude that most of them represent potential novel genes, with only a small fraction having protein-coding potential.     

WHEN DID YOU FIRST BEGIN TO FEEL IT? — LOCATING THE BEGINNING OF HUMAN CONSCIOUSNESS

In this paper we attempt to sharpen and to provide an answer to the question of when human beings first become conscious. Since it is relatively uncontentious that a capacity for raw sensation precedes and underpins all more sophisticated mental capacities, our question is tantamount to asking when human beings first have experiences with sensational content. Two interconnected features of our argument are crucial. First, we argue that experiences with sensational content are supervenient on facts about electrical activity in the cerebral cortex which can be ascertained through EEG readings. Second, we isolate from other notions of a'functioning brain'that which is required to underpin the view that a cortex is functioning in a way which could give rise to rudimentary conscious experiences. We investigate the development in the human fetus of the anatomical and chemical pathways which underpin (immature) cortical activity and the growth and maturation of the electrical circuitry specifically associated with sensational content in adult experience. We conclude (tentatively) that a fetus becomes conscious at about 30 to 35 weeks after conception; an answer based on a careful analysis of EEG readings at various stages of cortical development. Finally, we survey the possible ethical ramifications of our answer.     

Regionally Specific and Genome-Wide Analyses Conclusively Demonstrate the Absence of CpG Methylation in Human Mitochondrial DNA

Although CpG methylation clearly distributes genome-wide in vertebrate nuclear DNA, the state of methylation in the vertebrate mitochondrial genome has been unclear. Several recent reports using immunoprecipitation, mass spectrometry, and enzyme-linked immunosorbent assay methods concluded that human mitochondrial DNA (mtDNA) has much more than the 2 to 5% CpG methylation previously estimated. However, these methods do not provide information as to the sites or frequency of methylation at each CpG site. Here, we have used the more definitive bisulfite genomic sequencing method to examine CpG methylation in HCT116 human cells and primary human cells to independently answer these two questions. We found no evidence of CpG methylation at a biologically significant level in these regions of the human mitochondrial genome. Furthermore, unbiased next-generation sequencing of sodium bisulfite treated total DNA from HCT116 cells and analysis of genome-wide sodium bisulfite sequencing data sets from several other DNA sources confirmed this absence of CpG methylation in mtDNA. Based on our findings using regionally specific and genome-wide approaches with multiple human cell sources, we can definitively conclude that CpG methylation is absent in mtDNA. It is highly unlikely that CpG methylation plays any role in direct control of mitochondrial function.     

Absence of methylation at HpaII sites in three human genomic tRNA sequences.

It has been known since the development of nearest neighbor analysis that the frequency of the dinucleotide CpG is markedly suppressed in vertebrate DNA (i.e. less than %C x %G). This suppression appears to be heterogeneous since it was shown some years ago that three vertebrate tRNA genes did not exhibit CpG suppression. We have analyzed 13 different human tRNA genes and found that they also do not exhibit CpG suppression. Because CpG suppression has been linked, to some extent at least, to the methylation-deamination process by which a methylated CpG is mutated to TpG, we investigated whether the lack of suppression of CpG in tRNAs could originate from an absence of methylation. Three human tRNA genes were selected from Genbank (lysine, Proline, and Phenylalanine) and examined for methylation at HpaII sites by polymerase chain reaction (PCR) and Southern blot analysis. The observed patterns were consistent with the absence of methylation at the seven HpaII sites analyzed in and around the tRNA genes, and we predict that the remaining CpGs in these genes will be unmethylated. Since GC-rich promoter regions also escape CpG suppression and since they are generally unmethylated, avoidance of methylation may be a general explanation for the absence of CpG suppression in selected regions of vertebrate genomes.     

Inducible gene expression in transgenic Xenopus embryos

The amphibian Xenopus laevis has been successfully used for many years as a model system for studying vertebrate development. Because of technical limitations, however, molecular investigations have mainly concentrated on early stages. We have developed a straightforward method for stage-specific induction of gene expression in transgenic Xenopus embryos [1] [2]. This method is based on the Xenopus heat shock protein 70 (Xhsp70 [3]) promoter driving the expression of desired gene products. We found that ubiquitous expression of the transgene is induced upon relatively mild heat treatment. Green fluorescent protein (GFP) was used as a marker to monitor successful induction of gene expression in transgenic embryos. We used this method to study the stage specificity of Wnt signalling function. Transient ectopic Wnt-8 expression during early neurulation was sufficient to repress anterior head development and this capacity was restricted to early stages of neurulation. By transient over-expression at different stages of development, we show that frizzled-7 disrupted morphogenesis sequentially from anterior to posterior along the dorsal axis as development proceeds. These results demonstrate that this method for inducible gene expression in transgenic Xenopus embryos will be a very powerful tool for temporal analysis of gene function and for studying molecular mechanisms of vertebrate organogenesis.