A mutation that changes cell movement and cell fate in the zebrafish embryo

The study of developmental patterning has been facilitated by the availability of mutations that produce changes in cell fate, in animals such as Caenorhabditis elegans and Drosophila melanogaster. We now describe a zygotic lethal mutation in the zebrafish, Brachydanio rerio, that also changes how particular embryonic cells develop. Severe pattern deficiencies are observed that are restricted to a single body region, the trunk. The mutation may directly affect mesoderm, as somites do not form in the trunk. Head and tail structures, including tail somites, are relatively undisturbed. The earliest detected expression of the mutation is during gastrulation, when movements of mesodermal cells occur incorrectly. We injected prospective trunk mesodermal cells with lineage tracer dye and observed that in mutants these cells may enter a new body region, the tail, and there may express a new fate appropriate for the changed position.     

Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer

The mammalian Y chromosome acts as a dominant male determinant as a result of the action of a single gene, Sry, whose role in sex determination is to initiate testis rather than ovary development from early bipotential gonads. It does so by triggering the differentiation of Sertoli cells from supporting cell precursors, which would otherwise give follicle cells. The related autosomal gene Sox9 is also known from loss-of-function mutations in mice and humans to be essential for Sertoli cell differentiation; moreover, its abnormal expression in an XX gonad can lead to male development in the absence of Sry. These genetic data, together with the finding that Sox9 is upregulated in Sertoli cell precursors just after SRY expression begins, has led to the proposal that Sox9 could be directly regulated by SRY. However, the mechanism by which SRY action might affect Sox9 expression was not understood. Here we show that SRY binds to multiple elements within a Sox9 gonad-specific enhancer in mice, and that it does so along with steroidogenic factor 1 (SF1, encoded by the gene Nr5a1 (Sf1)), an orphan nuclear receptor. Mutation, co-transfection and sex-reversal studies all point to a feedforward, self-reinforcing pathway in which SF1 and SRY cooperatively upregulate Sox9 and then, together with SF1, SOX9 also binds to the enhancer to help maintain its own expression after that of SRY has ceased. Our results open up the field, permitting further characterization of the molecular mechanisms regulating sex determination and how they have evolved, as well as how they fail in cases of sex reversal.     

The cyclops mutation blocks specification of the floor plate of the zebrafish central nervous system

The floor plate is a set of epithelial cells present in the ventral midline of the neural tube in vertebrates that seems to have an important role in the developmental patterning of central nervous system fibre pathways, and arrangements of specific neurons. The floor plate arises from dorsal ectodermal cells closely associated with the mesoderm that forms notochord, and it may depend on interactions from the notochord for its specification. To learn the nature of these interactions we have analysed mutations in zebrafish (Brachydanio rerio). We report here that in wild-type embryos the floor plate develops as a simply organized single cell row, but that its development fails in embryos bearing the newly discovered zygotic lethal 'cyclops' mutation, cyc-1(b16). Mosaic analysis establishes that cyc-1 blocks floor plate development autonomously and reveals the presence of homeogenetic induction between floor plate cells.     

Tight linkage between a splicing mutation and a specific DNA haplotype in phenylketonuria

The first phenylketonuria mutation identified in the human phenylalanine hydroxylase gene is a single base substitution (GT----AT) in the canonical 5'-splice donor site of intron 12. Direct hybridization analysis using specific oligonucleotide probes demonstrates that the mutation is tightly associated with a specific restriction fragment-length polymorphism haplotype among mutant alleles. The splicing mutation is the most prevalent phenylketonuria allele among Caucasians, and the results suggest the possibility of detecting carriers of the genetic trait who have no family history of phenylketonuria.     

SCN1A基因4号外显子突变筛选

目的：探讨癫痫人群及正常人群SCN1A基因突变情况．方法：共采集癫痫病人血液181份、癫痫病人脑组织38份,正常人血液120份．提取基因组DNA,针对SCN1A基因4号外显子设计1对引物,进行聚合酶链反应（PCR）扩增,琼脂糖凝胶电泳,选取适合条件的PCR产物进行聚丙烯酰胺凝胶电泳,进行单链构象多态性分析,对个别PCR产物进行双向测序．结果：所有样本进行SCN1A的4号外显子筛选时,均未发现异常带出现．选取2例癫痫病人血液的PCR产物测序结果与基因组序列相比对,也未发现碱基改变．结论：癫痫是一种复杂综合征,研究的339癫痫患者中未发现SCN1A基因4号外显子突变．     

Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter

Defects in iron absorption and utilization lead to iron deficiency and overload disorders. Adult mammals absorb iron through the duodenum, whereas embryos obtain iron through placental transport. Iron uptake from the intestinal lumen through the apical surface of polarized duodenal enterocytes is mediated by the divalent metal transporter, DMTi. A second transporter has been postulated to export iron across the basolateral surface to the circulation. Here we have used positional cloning to identify the gene responsible for the hypochromic anaemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a multiple-transmembrane domain protein, expressed in the yolk sac, that is a candidate for the elusive iron exporter. Zebrafish ferroportin1 is required for the transport of iron from maternally derived yolk stores to the circulation and functions as an iron exporter when expressed in Xenopus oocytes. Human Ferroportin1 is found at the basal surface of placental syncytiotrophoblasts, suggesting that it also transports iron from mother to embryo. Mammalian Ferroportin1 is expressed at the basolateral surface of duodenal enterocytes and could export cellular iron into the circulation. We propose that Ferroportin1 function may be perturbed in mammalian disorders of iron deficiency or overload.     

斑马鱼胚胎在毒理学研究中的应用

斑马鱼胚胎对化学品暴露高度敏感,同时具有许多其他动物模型所没有的优势,因此,在毒理学研究中得到广泛应用。除化学品浓度外,在毒性检测中还需要考虑一系列因素的影响,如胚胎膜屏障作用、胚胎暴露时间、化学品的性状及不同化学品间的相互作用。目前,胚胎毒性评价方法由表型的观察逐渐向分子水平发展,更加侧重于对化学品作用机理的研究,使斑马鱼胚胎在毒性检测中具有更高特异性和灵敏性。     

Regulation of the G1-S transition in postembryonic neuronal precursors by axon ingrowth.

In the newly cellularized Drosophila embryo, progress through the cell cycle is regulated at the G2-M transition. We have examined cell-cycle regulation later in Drosophila development, in a group of postembryonic neuronal precursors. The S-phase precursor cells, which generate photoreceptor target neurons (lamina neurons) in the central nervous system, are not present in the absence of photoreceptor innervation. Here we report that axons selectively approach G1-phase precursors. Without axon ingrowth, lamina precursors do not enter their final S phase and by several criteria, arrest in the preceding G1 phase. These findings provide evidence that at this stage in development the control of cell division can occur at the G1-S transition.     

Alzheimer's disease. Molecular consequences of presenilin-1 mutation

Alzheimer's disease is characterized by accumulation in the brain of a family of insoluble amyloid peptides (Abeta peptides), which are produced as a result of the normal processing of beta-amyloid precursor protein (beta-APP). Russo et al. claim that a truncated Abeta peptide that lacks the first ten amino acids accumulates in the brains of patients carrying a mutant form of pre-senilin 1 (PS1), a protein that is involved in cleavage of beta-APP. However, we have found that this same species is also overrepresented in Alzheimer's patients with mutations in beta-APP itself. Our findings do not support the conclusion of Russo et al. that pathogenic PS1 mutations may control cleavage of beta-APP by beta-secretase.     

Nuclear transplantation in different strains of zebrafish

Single later blastula nuclei from AB strain of zebrafish (Danio rerio) were transplanted into enucleated unfertilized eggs of Long fin strain. Of 1119 cloning embryos, 14 reconstructed embryos developed into fry. DNA fingerprinting systems of the cloned fish were similar to those of the nuclear donor fish, but were distinctly different from those of the nuclear recipient fish. It confirmed that the genetic material originated from nuclear donor cell other than from nuclear recipient egg. The research suggested that the basic technique for nuclear transplantation performed with different strains of zebrafish has made a breakthrough. It should be helpful for the study of some important developmental problems such as gene function, the regulation ogene expression during animal development, the developmental potential of a nucleus and the interactions between the donor nucleus and the recipient cytoplasm, etc.     

A transforming mutation in the pleckstrin homology domain of AKT1 in cancer

Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.     

植物miRNAs前体的生物信息分析

miRNAs前体（pre-miRNAs）是产生成熟miRNAs的基因表达产物，能形成较为稳定的发卡环结构，具有较低的最小折叠自由能（minimal folding free cnergy，MFE）。在对植物pre-miRNAs长度、不同碱基含量、MFE、熵分析基础上，重点比较不同RNAs序列之间MFE与其长度的比值（MFEL）。结果表明MFEL是区分植物pre-miRNAs的一个很有效的参数：pre-miRNAs的MFEL值平均为-45．98kcal／mol，明显低于mRNAs（-23．08kcal／mol），tRNAs（-22．13kcal／mol）和rRNAs（-16．83kcal／mol）。使用MFEL参数可提高预测植物miRNAs基因的效率。     

植物miRNAs前体的生物信息分析

miRNAs前体（premiRNAs）是产生成熟miRNAs的基因表达产物,能形成较为稳定的发卡环结构,具有较低的最小折叠自由能（minimal folding free energy,MFE）。在对植物premiRNAs长度、不同碱基含量、MFE、熵分析基础上,重点比较不同RNAs序列之间MFE与其长度的比值（MFEL）。结果表明MFEL是区分植物premiRNAs的一个很有效的参数：premiRNAs的MFEL值平均为45.98 kcal/mol,明显低于mRNAs（23.08 kcal/     

Spontaneous forward mutation versus reversion frequencies for maize Adh1 in pollen

Reliable quantitative data on spontaneous, specific gene mutation frequencies in higher plants and animals are few. Detergents include low natural frequencies and difficulty in obtaining in excess of 10(6) scorable organisms or gametophytes. The alcohol dehydrogenase-1 gene (Adh1 gene; ADH enzyme EC 1.1.1.1.), as expressed in pollen grains, is among the exceptionably suitable; much is known about the maize ADHs, Adh1 function is totally dispensible in an aerobic environment and maize pollen is a trinucleate gametophyte which expresses much of its haploid genome, including Adh1. In particular, there have been two recent methodological advances. First, I am able to cytochemically stain pollen, before or after in vitro germination, for the presence of above 5% normal ADH activity. And second, ADH1- pollen grains survive allyl alcohol (C=C-C-OH) vapour concentrations which kill ADH1+ grains; this selection scheme was developed for yeast by Megnet. My genetic resolution is approximately one mutant (Adh1+-->ADH-) per 10(7) chemically selected, viable gametophytes, and one (phenotypic) revertant (Adh1--->ADH+) per 10(8) unselected gatetophytes. In this note, I compare spontaneous forward mutant frequency with previously published revertant frequencies for one naturally occurring and six ethyl methanesulphonate-induced Adhl-deficient (Adh1-) alleles.