mosaic eyes: a zebrafish gene required in pigmented epithelium for apical localization of retinal cell division and lamination

For proper function of the retina, the correct proportions of retinal cell types must be generated, they must be organized into cell-specific laminae, and appropriate synaptic connections must be made. To understand the genetic regulation of retinal development, we have analyzed mutations in the mosaic eyes gene that disrupt retinal lamination, the localization of retinal cell divisions to the retinal pigmented epithelial surface and retinal pigmented epithelial development. Although retinal organization is severely disrupted in mosaic eyes mutants, surprisingly, retinal cell differentiation occurs. The positions of dividing cells and neurons in the brain appear normal in mosaic eyes mutants, suggesting that wild-type mosaic eyes function is specifically required for normal retinal development. We demonstrate that mosaic eyes function is required within the retinal pigmented epithelium, rather than in dividing retinal cells. This analysis reveals an interaction between the retinal pigmented epithelium and the retina that is required for retinal patterning. We suggest that wild-type mosaic eyes function is required for the retinal pigmented epithelium to signal properly to the retina.     

Mutations affecting eye morphology in the developing zebrafish (Danio rerio)

The zebrafish (Danio rerio) has received considerable attention as a mainstream model for the molecular and genetic study of vertebrate development. In our laboratory, we have conducted a third-generation screen of chemically mutagenized zebrafish for recessive mutations affecting the visual system. This report describes the visible phenotypes and number of morphological mutants so far observed and presents a more detailed histological analysis of six of these mutations. Through analysis of mutant larvae, it was determined that several of the subtle morphological mutations resulted in degeneration of specific cellular layers of the retina. Other mutations resulted in some degeneration distributed diffusely across the entire retina or concentrated at the retinal margin. A single mutation affecting invagination of the optic cup and lens vesicle formation resulted in a failure to develop an anterior chamber. These results demonstrate the utility of a small-scale, highly focused screen for uncovering novel loci involved in retinal and eye development. Dev. Genet. 20:288–295, 1997. © 1997 Wiley-Liss, Inc.     

Mutations affecting retina development in Medaka

In a large scale mutagenesis screen of Medaka we identified 60 recessive zygotic mutations that affect retina development. Based on the onset and type of phenotypic abnormalities, the mutants were grouped into five categories: the first includes 11 mutants that are affected in neural plate and optic vesicle formation. The second group comprises 15 mutants that are impaired in optic vesicle growth. The third group includes 18 mutants that are affected in optic cup development. The fourth group contains 13 mutants with defects in retinal differentiation. 12 of these have smaller eyes, whereas one mutation results in enlarged eyes. The fifth group consists of three mutants with defects in retinal pigmentation. The collection of mutants will be used to address the molecular genetic mechanisms underlying vertebrate eye formation.     

Genetic dissection of the zebrafish retinal stem-cell compartment

In a large-scale forward-genetic screen, we discovered that a limited number of genes are required for the regulation of retinal stem cells after embryogenesis in zebrafish. In 18 mutants out of almost 2000 F2 families screened, the eye undergoes normal embryonic development, but fails to continue growth from the ciliary marginal zone (CMZ), the post-embryonic stem-cell niche. Class I-A mutants (5 loci) display lower amounts of proliferation in the CMZ, while nearly all cells in the retina appear differentiated. Class I-B mutants (2 loci) have a reduced CMZ with a concomitant expansion in the retinal pigmented epithelium (RPE), suggesting a common post-embryonic stem cell is the source for these neighboring cell types. Class II encompasses three distinct types of mutants (11 loci) with expanded CMZ, in which the progenitor population is arrested in the cell cycle. We also show that in at least one combination, the reduced CMZ phenotype is genetically epistatic to the expanded CMZ phenotype, suggesting that Class I genes are more likely to affect the stem cells and Class II the progenitor cells. Finally, a comparative mapping analysis demonstrates that the new genes isolated do not correspond to genes previously implicated in stem-cell regulation. Our study suggests that embryonic and post-embryonic stem cells utilize separable genetic programs in the zebrafish retina.     

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.     

Isolation,characterization and mapping of temperature-sensitive mutants of mycobacteriophage I3

Eighteen temperature-sensitive mutants of mycobacteriophage I3 have been isolated and partially characterized. All the mutants were defective in vegetative replication. Based on temperature shift experiments with the temperature sensitive mutants, the thermosensitive phase of the phage development period has been characterized for each mutant. The genes have been mapped by recombination analysis. The early, continuous and middle genes seem to cluster on the genetic map     

Leaf-variegated mutations and their responsible genes in Arabidopsis thaliana

Leaf variegation has long been known as a recessive genetic trait in higher plants. Unlike albino mutants, leaf-variegated mutants are non-lethal and thus enable us to study a novel mechanism of plastid development and maintenance. Variegation results from a defect that makes chloroplast development unstable, since at least part of the tissues gives rise to normal chloroplasts. Despite the fact that leaf-variegated mutants have contributed to the findings of maternal inheritance or have been used as genetic markers, these mutations and the responsible loci have been poorly understood at the molecular level. A comprehensive study of the leaf-variegated mutants is possible in Arabidopsis, since such mutants have been known and the cloning can be at relative ease as a model plant. Here I summarize recent progress on characterization of the Arabidopsis leaf-variegated mutants. Detailed analysis of the responsible loci revealed that variegation is caused by a defect in various metabolic pathways related to organelle functions. Thus, studies on these genes provide us with novel redundant mechanisms by which heteroplasmic organelles such as plastids and mitochondria can survive from an environmental stress.     

The cell cycle and retinal histogenesis fidget mutant mice.

The autoradiographic method using [³H]thymidine has shown that the autosomal recessive mutant gene fidget (gene symbol fi) prolonging the presynthetic period of the cell cycle in the retinal anlage in homozygotes retards the transition of retinal cells to the differentiated state. Some retinal cells of normal embryos (+/+) start their transition to the differentiated state on the 11th day of embryogenesis, while in $\text{fi}\text{fi}$ embryos this process starts only on the 12th day. An active transition of retinal cells to the differentiated state especially in the peripheral zone of the mutant retina takes place 2 days later as compared to normal embryos. The number of differentiating cells in the retina of mutants at the stages of development studied is considerably lower as compared to the norm. The analysis of the cell cycle parameters in 15-day embryos has shown that in the mutants the retina is less mature as compared to +/+ embryos. The sequence of transition of various cell types to the differentiated state in the retina of $\text{fi}\text{fi}$ embryos is the same as in the norm. Gene fidget seems to interfere with proliferative rather than critical (quantal) cell cycles in the developing mouse retina.     

Alteration of neural tissue structure by expression of polysialic acid induced by viral delivery of PST polysialyltransferase

The expression of polysialic acid (PSA) on neural cell adhesion molecule (NCAM) is known to attenuate cell-cell interactions. During neural development the widespread expression of PSA-NCAM creates permissive conditions for the migration of neuronal and glial precursors and the guidance and targeting of axons. NCAM polysialylation can occur via either of two specific sialyltransferases, ST8SiaII (STX) and ST8SiaIV (PST), and the purpose of this study was to determine if retroviral delivery of either PST or STX could induce PSA expression in vivo and thereby alter tissue plasticity. Retroviruses expressing GFP-PST or GFP-STX were injected into embryonic retina, and development was evaluated by examining neuroepithelial structure, the expression of markers for specific cell types, cellular proliferation, and apoptosis. Chick retina was chosen because it down-regulates PSA early in its development and has a highly stereotyped program of morphogenesis. Retroviral expression of PST induced PSA expression in retina and resulted in severe but localized alterations in retinal morphogenesis, including an early disruption of radial glial cell morphology, highly disorganized retinal layers, and invasion of pigmented cells into the neural retina. In contrast, retroviral delivery of STX did not induce PSA expression or affect morphogenesis. These findings demonstrate that expression of PSA is sufficient to promote morphological alterations in a relatively nonplastic neural tissue.     

Mutations in laminin alpha 1 result in complex, lens-independent ocular phenotypes in zebrafish

We report phenotypic and genetic analyses of a recessive, larval lethal zebrafish mutant, bal(a69), characterized by severe eye defects and shortened body axis. The bal(a69) mutation was mapped to chromosome 24 near the laminin alpha 1 (lama1) gene. We analyzed the lama1 gene sequence within bal(a69) embryos and two allelic mutants, bal(arl) and bal(uw1). Missense (bal(a69)), nonsense (bal(arl)), and frameshift (bal(uw1)) alterations in lama1 were found to underlie the phenotypes. Extended analysis of bal(a69) ocular features revealed disrupted lens development with subsequent lens degeneration, focal cornea dysplasia, and hyaloid vasculature defects. Within the neural retina, the ganglion cells showed axonal projection defects and ectopic photoreceptor cells were noted at inner retinal locations. To address whether ocular anomalies were secondary to defects in lens differentiation, bal(a69) mutants were compared to embryos in which the lens vesicle was surgically removed. Our analysis suggests that many of the anterior and posterior ocular defects in bal(a69) are independent of the lens degeneration. Analysis of components of focal adhesion signaling complexes suggests that reduced focal adhesion kinase activation underlies the anterior segment dysgenesis in lama1 mutants. To assess adult ocular phenotypes associated with lama1 mutations, genetic mosaics were generated by transplanting labeled bal cells into ocular-fated regions of wild-type blastulas. Adult chimeric eyes displayed a range of defects including anterior segment dysgenesis and cataracts. Our analysis provides mechanistic insights into the developmental defects and ocular pathogenesis caused by mutations in laminin subunits.     

Identification and genetic mapping of four novel genes that regulate leaf development in Arabidopsis

Molecular and genetic characterizations of mutants have led to a better understanding of many developmental processes in the model system Arabidopsis thaliana.However,the leaf development that is specific to plants has been little studies.With the aim of contributing to the genetic dissection of leaf development,we have performed a large-scare screening for mutants with abnormal leaves.Among a great number of leaf mutants we have generated by T-DNA and transposon tagging and ethylmethae sulfonate (EMS) mutagenesis,four independent mutant lines have been identified and studied genetically.Phenotypes of these mutant lines represent the defects of four novel muclear genes designated LL1(LOTUS LEAF 1),LL2(LOTUS LEAF2),URO(UPRIGHT ROSETTE),and EIL(ENVIRONMENT CONDITION INDUCED LESION).The phenotypic analysis indicates that these genes play important roles during leaf development.For the further genetic analysis of these genes and the map-based cloning of LL1 and LL2,we have mapped these genes to chromosome regions with an efficient and rapid mapping method.     

Distinct developmental programs require different levels of Bmp signaling during mouse retinal development

The Bmp family of secreted signaling molecules is implicated in multiple aspects of embryonic development. However, the cell-type-specific requirements for this signaling pathway are often obscure in the context of complex embryonic tissue interactions. To define the cell-autonomous requirements for Bmp signaling, we have used a Cre-loxP strategy to delete Bmp receptor function specifically within the developing mouse retina. Disruption of a Bmp type I receptor gene, Bmpr1a, leads to no detectable eye abnormality. Further reduction of Bmp receptor activity by removing one functional copy of another Bmp type I receptor gene, Bmpr1b, in the retina-specific Bmpr1a mutant background, results in abnormal retinal dorsoventral patterning. Double mutants completely lacking both of these genes exhibit severe eye defects characterized by reduced growth of embryonic retina and failure of retinal neurogenesis. These studies provide direct genetic evidence that Bmpr1a and Bmpr1b play redundant roles during retinal development, and that different threshold levels of Bmp signaling regulate distinct developmental programs such as patterning, growth and differentiation of the retina.     

The identification of candidate genes for a reverse genetic analysis of development and function in the Arabidopsis gynoecium

The screening for mutants and their subsequent molecular analysis has permitted the identification of a number of genes of Arabidopsis involved in the development and functions of the gynoecium. However, these processes remain far from completely understood. It is clear that in many cases, genetic redundancy and other factors can limit the efficiency of classical mutant screening. We have taken the alternative approach of a reverse genetic analysis of gene function in the Arabidopsis gynoecium. A high-throughput fluorescent differential display screen performed between two Arabidopsis floral homeotic mutants has permitted the identification of a number of genes that are specifically or preferentially expressed in the gynoecium. Here, we present the results of this screen and a detailed characterization of the expression profiles of the genes identified. Our expression analysis makes novel use of several Arabidopsis floral homeotic mutants to provide floral organ-specific gene expression profiles. The results of these studies permit the efficient targeting of effort into a functional analysis of gynoecium-expressed genes.     

Genetic Control of Root Hair Development in Arabidopsis thaliana

Visual examination of roots from 12,000 mutagenized Arabidopsis seedlings has led to the identification of more than 40 mutants impaired in root hair morphogenesis. Mutants from four phenotypic classes have been characterized in detail, and genetic tests show that these result from single nuclear recessive mutations in four different genes designated RHD1, RHD2, RHD3, and RHD4. The phenotypic analysis of the mutants and homozygous double mutants has led to a proposed model for root hair development and the stages at which the genes are normally required. The RHD1 gene product appears to be necessary for proper initiation of root hairs, whereas the RHD2, RHD3, and RHD4 gene products are required for normal hair elongation. These results demonstrate that root hair development in Arabidopsis is amenable to genetic dissection and should prove to be a useful model system to study the molecular mechanisms governing cell differentiation in plants.     

拟南芥雄性不育突变体ms1502的遗传及定位分析

通过EMS诱变、背景纯化与遗传分析,从拟南芥（Arabidopsis thaliana）中筛选到了一棵隐性单基因控制的雄性不育突变体ms1502。细胞学观察发现,突变体在小孢子从四分体释放出后花药绒毡层过早衰亡,小孢子的内容物不正常地凝聚,最终无法形成正常的花粉粒。利用图位克隆的方法对该基因MSl502进行了定位,结果表明MS1502位于第4条染色体上分子标记F25124和T12H20之间105kb区间内。目前该区间内尚未见到花药发育必需基因（不育基因）的报道,因此MS1502是一个控制花粉发育的新基因。     

Goethe and the ABC model of flower development.

About 10 years ago, the ABC model for the genetic control of flower development was proposed. This model was initially based on the analysis of mutant flowers but has subsequently been confirmed by molecular analysis. This paper describes the 200-year history behind this model, from the late 18th century when Goethe arrived at his idea of plant metamorphosis, to the genetic studies on flower mutants carried out on Arabidopsis and Antirrhinum in the late 20th century.     

A novel 'gene insertion/marker out' (GIMO) method for transgene expression and gene complementation in rodent malaria parasites

Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel 'gene insertion/marker out' (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research.     

The genetics of phytochrome signalling in Arabidopsis

The application of Arabidopsis genetics to research into the responses of plants to light has enabled rapid recent advances in this field. The plant photoreceptor phytochrome mediates well-defined responses that can be exploited to provide elegant and specific genetic screens. By this means, not only have mutants affecting the phytochromes themselves been isolated, but also mutants affecting the transduction of phytochrome signals. The genes involved in these processes have now begun to be characterized by using this genetic approach to isolate signal transduction components. Most of the components characterized so far are capable of being translocated to the cell nucleus, and they may help to define a new system of regulation of gene expression. This review summarises the ongoing contribution made by genetics to our understanding of light perception and signal transduction by the phytochrome system.