果蝇睾丸基因敲除突变体的构建及表型分析

生殖系统功能的正常维持是物种繁衍的基础,需要多基因协同作用,但其中许多基因的具体功能和作用机制并不清楚。本研究选取了果蝇(Drosophila melanogaster)中8个在睾丸中表达、功能未知且与人(Homo sapiens)和小鼠(Mus musculus)高度同源的基因(CG4161、CG11475、CG2921、CG10541、CG7276、CG3800、CG8117和CG16779),分析了它们在不同组织中的表达水平,并分别检测了它们在雄性生殖系统中的功能。在这8个基因中,前5个为睾丸优势表达基因,其余3个为全身性表达。首先,利用CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)技术结合同源定向修复(homology-directed repair, HDR)在果蝇中对8个候选基因逐一进行敲除,建立了纯合的基因敲除突变品系;然后对这些品系的雄蝇进行了生育力测试及睾丸细胞学观察。结果显示,CG7276和CG3800基因敲除果蝇出现部分雄蝇不育且可育雄蝇后代数量较野生型显著下降。睾丸解剖观察显示CG7276和CG3800的功能缺失可导致雄蝇分别出现不同程度的精囊缩小及精原干细胞减少和细胞分布混乱;染色结果也提示CG7276和CG3800在精子的成熟过程中发挥一定作用。其他6个基因突变并未导致雄蝇育性变化或睾丸形态异常。这些突变体的获得及表型的初步分析为进一步研究基因功能及机制提供了良好的动物模型及基础。     

From Haeckel to event-pairing: the evolution of developmental sequences

Summary Development involves a series of developmental events, separated by transformations, that follow a particular order or developmental sequence. The sequence may in turn be arbitrarily subdivided into contiguous segments (developmental stages). We discuss the properties of developmental sequences. We also examine the differing analytical approaches that have been used to analyse developmental sequences in an evolutionary context. Ernst Haeckel was a pioneer in this field. His approach was evolutionary and he introduced the idea of sequence heterochrony (evolutionary changes in the sequence of developmental events). Despite the availability of detailed developmental data (e.g. Franz Keibel’s ‘Normal Tables’), Haeckel was unable to undertake a quantitative analysis of developmental data. This is now possible through computer-based analytical techniques such as event-pairing, which can extract important biological information from developmental sequences by mapping them onto established phylogenies. It may also yield data that can be used in phylogeny reconstruction, although the inherent ‘non-independence’ of the data may make this invalid. In future, the methods discussed here may be applied to the analysis of patterns of gene expression in embryos, or adapted to studying gene order on chromosomes.     

From individuals to aggregations: the interplay between behavior and physics

This paper analyses the processes by which organisms form groups and how social forces interact with environmental variability and transport. For aquatic organisms, the latter is especially important-will sheared or turbulent flows disrupt organism groups? To analyse such problems, we use individual-based models to study the environmental and social forces leading to grouping. The models are then embedded in turbulent flow fields to gain an understanding of the interplay between the forces acting on the individuals and the transport induced by the fluid motion. Instead of disruption of groups, we find that flows often enhance grouping by increasing the encounter rate among groups and thereby promoting merger into larger groups; the effect breaks down for strong flows. We discuss the transformation of individual-based models into continuum models for the density of organisms. A number of subtle difficulties arise in this process; however, we find that a direct comparison between the individual model and the continuum model is quite favorable. Finally, we examine the dynamics of group statistics and give an example of building an equation for the spatial and temporal variations of the group-size distribution from the individual-based simulations. These studies lay the groundwork for incorporating the effects of grouping into models of the large scale distributions of organisms as well as for examining the evolutionary consequences of group formation.     

From phenotype to genotype: a Bayesian solution

The study of biological systems commonly depends on inferring the state of a 'hidden' variable, such as an underlying genotype, from that of an 'observed' variable, such as an expressed phenotype. However, this cannot be achieved using traditional quantitative methods when more than one genetic mechanism exists for a single observable phenotype. Using a novel latent class Bayesian model, it is possible to infer the prevalence of different genetic elements in a population given a sample of phenotypes. As an exemplar, data comprising phenotypic resistance to six antimicrobials obtained from passive surveillance of Salmonella Typhimurium DT104 are analysed to infer the prevalence of individual resistance genes, as well as the prevalence of a genomic island known as SGI1 and its variants. Three competing models are fitted to the data and distinguished between using posterior predictive p-values to assess their ability to predict the observed number of unique phenotypes. The results suggest that several SGI1 variants circulate in a few fixed forms through the population from which our data were derived. The methods presented could be applied to other types of phenotypic data, and represent a useful and generic mechanism of inferring the genetic population structure of organisms.     

Mimicry: developmental genes that contribute to speciation

Despite renewed interest in the role of natural selection as a catalyst for the origin of species, the developmental and genetic basis of speciation remains poorly understood. Here we describe the genetics of Müllerian mimicry in Heliconius cydno and H. melpomene (Lepidoptera: Nymphalidae), sister species that recently diverged to mimic other Heliconius. This mimetic shift was a key step in their speciation, leading to pre- and postmating isolation. We identify 10 autosomal loci, half of which have major effects. At least eight appear to be homologous with genes known to control pattern differences within each species. Dominance has evolved under the influence of identifiable "modifier" loci rather than being a fixed characteristic of each locus. Epistasis is found at many levels: phenotypic interaction between specific pairs of genes, developmental canalization due to polygenic modifiers so that patterns are less sharply defined in hybrids, and overall fitness through ecological selection against nonmimetic hybrid genotypes. Most of the loci are clustered into two genomic regions or "supergenes," suggesting color pattern evolution is constrained by preexisting linked elements that may have arisen via tandem duplication rather than having been assembled by natural selection. Linkage, modifiers, and epistasis affect the strength of mimicry as a barrier to gene flow between these naturally hybridizing species and may permit introgression in genomic regions unlinked to those under disruptive selection. Müllerian mimics in Heliconius use different genetic architectures to achieve the same mimetic patterns, implying few developmental constraints. Therefore, although developmental and genomic constraints undoubtedly influence the evolutionary process, their effects are probably not strong in comparison with natural selection.     

Pancreatic beta-cells: From generation to regeneration

The pancreas is composed of two main compartments consisting of endocrine and exocrine tissues. The majority of the organ is exocrine and responsible for the synthesis of digestive enzymes and for their transport via an intricate ductal system into the duodenum. The endocrine tissue represents less than 2% of the organ and is organized into functional units called islets of Langerhans, comprising alpha-, beta-, delta-, epsilon- and PP-cells, producing the hormones glucagon, insulin, somatostatin, ghrelin and pancreatic polypeptide (PP), respectively. Insulin-producing beta-cells play a central role in the control of the glucose homeostasis. Accordingly, absolute or relative deficiency in beta-cells may ultimately lead to type 1 and/or type 2 diabetes, respectively. One major goal of diabetes research is therefore to understand the molecular mechanisms controlling the development of beta-cells during pancreas morphogenesis, but also those underlying the regeneration of adult injured pancreas, and assess their significance for future cell-based therapy. In this review, we will therefore present new insights into beta-cell development with focus on beta-cell regeneration.     

From cell fates to morphology: developmental genetics of the Caenorhabditis elegans male tail.

The C. elegans male tail is being studied as a model to understand how genes specify the form of multicellular animals. Morphogenesis of the specialized male copulatory organ takes place in the last larval stages during male development. Genetic analysis is facilitated because the structure is not necessary for male viability or for strain propagation. Analysis of developmental mutants, isolated in several functional and morphological screens, has begun to reveal how fates of cells are determined in the cell lineages, and how the specification of cell fates affects the morphology of the structure. Cytological studies in wild type and in mutants have been used to study the mechanism of pattern formation in the tail peripheral nervous system. The ultimate goal is to define the entire pathway leading to the male copulatory organ.     

PGMapper: a web-based tool linking phenotype to genes

SUMMARY: With the availability of whole genome sequence in many species, linkage analysis, positional cloning and microarray are gradually becoming powerful tools for investigating the links between phenotype and genotype or genes. However, in these methods, causative genes underlying a quantitative trait locus, or a disease, are usually located within a large genomic region or a large set of genes. Examining the function of every gene is very time consuming and needs to retrieve and integrate the information from multiple databases or genome resources. PGMapper is a software tool for automatically matching phenotype to genes from a defined genome region or a group of given genes by combining the mapping information from the Ensembl database and gene function information from the OMIM and PubMed databases. PGMapper is currently available for candidate gene search of human, mouse, rat, zebrafish and 12 other species. AVAILABILITY: Available online at http://www.genediscovery.org/pgmapper/index.jsp.     

From genotype to phenotype: buffering mechanisms and the storage of genetic information

DNA sequence variation is abundant in wild populations. While molecular biologists use genetically homogeneous strains of model organisms to avoid this variation, evolutionary biologists embrace genetic variation as the material of evolution since heritable differences in fitness drive evolutionary change. Yet, the relationship between the phenotypic variation affecting fitness and the genotypic variation producing it is complex. Genetic buffering mechanisms modify this relationship by concealing the effects of genetic and environmental variation on phenotype. Genetic buffering allows the build-up and storage of genetic variation in phenotypically normal populations. When buffering breaks down, thresholds governing the expression of previously silent variation are crossed. At these thresholds, phenotypic differences suddenly appear and are available for selection. Thus, buffering mechanisms modulate evolution and regulate a balance between evolutionary stasis and change. Recent work provides a glimpse of the molecular details governing some types of genetic buffering.     

BRAF gene: From human cancers to developmental syndromes

The BRAF gene encodes for a serine/threonine protein kinase that participates in the MAPK/ERK signalling pathway and plays a vital role in cancers and developmental syndromes (RASopathies). The current review discusses the clinical significance of the BRAF gene and other members of RAS/RAF cascade in human cancers and RAS/MAPK syndromes, and focuses the molecular basis and clinical genetics of BRAF to better understand its parallel involvement in both tumourigenesis and RAS/MAPK syndromes—Noonan syndrome, cardio-facio-cutaneous syndrome and LEOPARD syndrome.     

From temporal processing to developmental language disorders: mind the gap

The ‘rapid temporal processing’ and the ‘temporal sampling framework’ hypotheses have been proposed to account for the deficits in language and literacy development seen in specific language impairment and dyslexia. This paper reviews these hypotheses and concludes that the proposed causal chains between the presumed auditory processing deficits and the observed behavioural manifestation of the disorders are vague and not well established empirically. Several problems and limitations are identified. Most data concern correlations between distantly related tasks, and there is considerable heterogeneity and variability in performance as well as concerns about reliability and validity. Little attention is paid to the distinction between ostensibly perceptual and metalinguistic tasks or between implicit and explicit modes of performance, yet measures are assumed to be pure indicators of underlying processes or representations. The possibility that diagnostic categories do not refer to causally and behaviourally homogeneous groups needs to be taken seriously, taking into account genetic and neurodevelopmental studies to construct multiple-risk models. To make progress in the field, cognitive models of each task must be specified, including performance domains that are predicted to be deficient versus intact, testing multiple indicators of latent constructs and demonstrating construct reliability and validity.     

From genotype to phenotype: linking bioinformatics and medical informatics ontologies

A small group of around 40 people came together at the Chancellors Conference Centre in Manchester for the Ontologies Workshop, chaired by Alan Rector and Robert Stevens. The workshop was, rather strangely, spread over 2 half days. In hindsight, this programme worked very well as it gave people the opportunity to chat over a drink on the Saturday evening and share ideas, before launching into the second half on the following day. The participants were from various walks of life, all with a common interest in finding out more about ontologies and promoting collaborations between the medical informatics and bioinformatics ontology communities.