Mapping genes essential for embryo development in Arabidopsis thaliana.

Summary We have previously isolated and characterized over 90 recessive mutants of Arabidopsis thaliana defective in embryo development. These emb mutants have been shown to differ in lethal phase, extent of abnormal development, and response in culture. We demonstrate in this report the value and efficiency of mapping emb genes relative to visible and molecular markers. Sixteen genes essential for embryo development were mapped relative to visible markers by analyzing progeny of selfed F₁ plants. Embryonic lethals are now the most common type of visible marker included on the linkage map of Arabidopsis. Backcrosses were used in several cases to orient genes relative to adjacent markers. Three genes were located to chromosome arms with telotrisomics by screening for a reduction in the percentage of aborted seeds produced by F₁ plants. A restriction fragment length polymorphism (RFLP) mapping strategy that utilizes pooled EMB/EMB F₂ plants was devised to increase the efficiency of mapping embryonic lethals relative to molecular markers. This strategy was tested by demonstrating that the biol locus of Arabidopsis is within 0.5 cM of an existing RFLP marker. Mapping embryonic lethals with both visible and molecular markers may therefore help to identify large numbers of genes with essential functions in Arabidopsis.     

An uncoupling screen for autonomous embryo mutants in Arabidopsis thaliana

Simple de novo screens in Arabidopsis thaliana have previously identified mutants that affect endosperm development but viable-embryo mutants have not been identified. Our strategy to identify autonomous embryo development was to uncouple embryo and endosperm fertilisation. This involved a male-sterile mutant population being crossed with a distinct pollen parent—the pollen was needed to initiate endosperm development and because it was distinct, the maternal progeny could be selected from the hybrid population. This process was refined over three stages, resulting in a viable approach to screen for autonomous embryo mutants. From 8,000 screened plants, a mutation was isolated in which the integument cells extended from the ovule and proliferated into a second complete twinned ovule. Some embryos from the mutant were normal but others developed fused cotyledons. In addition, a proportion of the progeny lacked paternal genes.     

In vitro morphogenesis of arrested embryos from lethal mutants of Arabidopsis thaliana

Summary Arrested embryos from lethal (emb) mutants of Arabidopsis thaliana were rescued on a nutrient medium designed to promote plant regeneration from immature wild-type cotyledons. The best response was observed with mutant embryos arrested at the heart to cotyledon stages of development. Embryos arrested at a globular stage produced callus but failed to turn green or form normal shoots in culture. Many of the mutant plants produced in culture were unusually pale with abnormal leaves, rosettes, and patterns of reproductive development. Other plants were phenotypically normal except for the presence of siliques containing 100% aborted seeds following self-pollination. These results demonstrate that genes with essential functions during plant embryo development differ in their pattern of expression at later stages of the life cycle. Most of the 15 genes examined in this study were essential for embryogenesis but were required again for subsequent stages of development. Only EMB24 appeared to be limited in function to embryo development. These differences in the response of mutant embryos in culture may facilitate the classification of embryonic lethals and the identification of genes with developmental rather than housekeeping functions.     

gurke and pasticcino3 mutants affected in embryo development are impaired in acetyl-CoA carboxylase

Normal embryo development is required for correct seedling formation. The Arabidopsis gurke and pasticcino3 mutants were isolated from different developmental screens and the corresponding embryos exhibit severe defects in their apical region, affecting bilateral symmetry. We have recently identified lethal acc1 mutants affected in acetyl-CoA carboxylase 1 (ACCase 1) that display a similar embryo phenotype. A series of crosses showed that gk and pas3 are allelic to acc1 mutants, and direct sequencing of the ACC1 gene revealed point mutations in these new alleles. The isolation of leaky acc1 alleles demonstrated that ACCase 1 is essential for correct plant development and that mutations in ACCase affect cellular division in plants, as is the case in yeast. Interestingly, significant metabolic complementation of the mutant phenotype was obtained by exogenous supply of malonate, suggesting that the lack of cytosolic malonyl-CoA is likely to be the initial factor leading to abnormal development in the acc1 mutants.     

Isolation and Characterization of 51 embryo-specific Mutations of Maize

A plant embryo consists of an embryonic axis, which eventually grows into the adult body, and one or two nutritive structures, the cotyledons. In the grasses embryo morphogenesis can be divided into three periods: during the first the embryo is regionalized into an embryo proper and suspensor, during the second the embryonic axis is established, and during the third vegetative structures are elaborated. Maize, with its well-characterized embryo-genesis, powerful genetics, and transposon tagging stocks, offers an attractive system for mutational analysis of these events. We have isolated 51 embryo-specific (emb) mutations from active Robertson's Mutator maize stocks. These are single-gene recessive lethals that represent at least 45 independent mutation events. Each of the 25 mutations was located to a chromosome arm using a B-A translocation set that uncovers approximately 40% of the genome; the same test failed to locate 20 others. The embryo phenotype of 27 mutations was characterized by examining mature mutant embryos in fresh dissection: the various emb mutations differ in phenotype and each is consistent in its expression. All 27 mutations result in retarded embryos that are morphologically abnormal. Nine mutants are blocked during the first period; 10 mutants are blocked during the second period; and eight mutants are blocked during the third period. Based on both the genetic and developmental data, it is likely that there are many loci that can mutate to give the emb phenotype and that these genes are crucial to the morphogenesis of the embryo.     

Lethals, Steriles and Deficiencies in a Region of the X Chromosome of CAENORHABDITIS ELEGANS

Twenty-one X-linked recessive lethal and sterile mutations balanced by an unlinked X-chromosome duplication have been identified following EMS treatment of the small nematode, Caenorhabditis elegans. The mutations have been assigned by complementation analysis to 14 genes, four of which have more than one mutant allele. Four mutants, all alleles, are temperature-sensitive embryonic lethals. Twelve mutants, in ten genes, are early larval lethals. Two mutants are late larval lethals, and the expression of one of these is influenced by the number of X chromosomes in the genotype. Two mutants are maternal-effect lethals; for both, oocytes made by mutant hermaphrodites are rescuable by wild-type sperm. One of the maternal-effect lethals and two larval lethals are allelic. One mutant makes defective sperm. The lethals and steriles have been mapped by recombination and by complementation testing against 19 deficiencies identified after X-ray treatment. The deficiencies divide the region, about 15% of the X-chromosome linkage map, into at least nine segments. The deficiencies have also been used to check the phenotypes of hemizygous lethal and sterile hermaphrodites.     

Genetic Analysis of Two Allelic Temperature-Sensitive Mutants of DROSOPHILA MELANOGASTER Both of Which Are Zygotic and Maternal-Effect Lethals

After fertilization, the development of a zygote depends upon both gene products synthesized by its maternal parent and gene products synthesized by the zygote itself. To analyze genetically the relative contributions of these two sources of gene products, several laboratories have been isolating two classes of mutants of Drosophila melanogaster: maternal-effect lethals and zygotic lethals. This report concerns the analysis of two temperature-sensitive mutants, OX736hs and PC025hs, which were isolated as alleles of a small-disc mutant, l(3)1902. These alleles are not only zygotic lethals, but also maternal-effect lethals. They have temperature-sensitive periods during larval life and during oogenesis. Mutant larvae exposed continuously to restrictive temperature have small discs. One-or two-day exposures to the restrictive temperature administered during the third larval instar lead to a homeotic transformation of the midlegs and hindlegs to the pattern characteristic of the forelegs. Mutant females exposed to the restrictive temperature during oogenesis produce eggs that can develop until gastrulation, but do not hatch.--The existence of these mutants, and one that was recently described by another group, implies that there may be a class of genes, heretofore unrecognized, whose products are synthesized during oogenesis, are essential for embryogenesis and are also synthesized during larval stages within imaginal disc cells.     

Developmental profiles of three embryo-lethal maize mutants lacking leaf primordia: ptd*-1130, cp*-1418, and bno*-747B

The defective kernel (dek) mutants of maize are altered in both their embryo and endosperm development. Earlier studies have indicated that some of the dek mutants are unable to form shoot apical meristems or leaf primoirda. We have examined three embryo lethal dek mutants of this type, ptd*-1130, cp*-1418, and bno*-747B, to obtain a developmental profile for each. Allelism tests show that these three mutants are not allelic. Embryos were examined in early, mid-, and late kernel development as well as at kernel maturity by dissection and sectioning procedures and also at kernel maturity by scanning electron microscopy. All three mutants lag behind normal embryos in their rate of development. Embryos of ptd*-1130 reached the transition stage by early kernel development and progressed no further but underwent cell enlargement and necrosis during late kernel development. Embryos of cp*-1418 reached an early coleoptilar stage by midkernel development. They subsequently increased in size but did not form any leaf primordia. At kernel maturity, they no longer had a shoot apical meristem but often had a well formed root meristem. They appeared to remain healthy and did not become necrotic. Embryos of bno*747B reached the early coleoptilar stage by early kernel development but progressed no further. By kernel maturity, they had grown into masses of irregularly shaped embryonic tissue that no longer resembled any normal embryo stage but were not necrotic. None of these three mutants responded to attempts to support continued embryo development when cultured, but all three mutants formed callus on N6 and MS media supplemented with 2,4-D. These results indicate that these mutants are all uniformly blocked at specific stages early in embryonic development, have different subsequent developmental fates, and represent three different genes performing unique functions that are essential for embryogenesis.     

Genetic characterization and floral development of female sterile and stubby head, two aposporous mutants of pearl millet

 Apomixis has never been reported in natural populations of pearl millet [Pennisetum glaucum (L.) R.Br.], although many wild relatives of pearl millet are obligate or facultative aposporous apomicts. Four-nucleate aposporous embryo sacs are formed from somatic cells of the nucellus that do not undergo meiosis. Two mutants of pearl millet, female sterile (fs) and stubby head, have two developmental characteristics in common: a significant reduction in head length compared with the wild-type and the formation of aposporous embryo sacs. Reproductive development in fs and stubby head mutants was examined in depth because of the potential for illuminating basic cellular or developmental factors that may function to alter embryo sac development. Genetic analysis of stubby head showed that this phenotype is conferred by genes at two loci linked in coupling within 29 cM. Crosses between fs and stubby head mutants showed that, despite the similarities in phenotypes, the mutations are at different loci. The mutants differ from wild-type in their inflorescence structure from the time of initiation of spikelet primordia through terminal differentiation of the ovule. Both mutations could be categorized as meristic, since a change in inflorescence branch or organ number was common and gynoecium development varied. We speculate that heterochronic development of the floral meristem and organ initiation/specification programs may be the underlying mechanism for phenotypic changes in these mutants throughout the floral phase. Received: 25 October 1996 / Accepted: 13 March 1997     

Deletion of core components of the plastid protein import machinery causes differential arrest of embryo development in Arabidopsis thaliana

Among the genes that have recently been pinpointed to be essential for plant embryo development a large number encodes plastid proteins suggesting that embryogenesis is linked to plastid localized processes. However, nuclear encoded plastid proteins are synthesized as precursors in the cytosol and subsequently have to be transported across the plastid envelopes by a complex import machinery. We supposed that deletion of components of this machinery should allow a more general assessment of the role of plastids in embryogenesis since it will not only affect single proteins but instead inhibit the accumulation of most plastid proteins. Here we have characterized three Arabidopsis thaliana mutants lacking core components of the Toc complex, the protein translocase in the outer plastid envelope membrane, which indeed show embryo lethal phenotypes. Remarkably, embryo development in the atToc75-III mutant, lacking the pore forming component of the translocase, was arrested extremely early at the two-cell stage. In contrast, despite the complete or almost complete lack of the import receptors Toc34 and Toc159, embryo development in the a tToc33/34 and atToc132/159 mutants proceeded slowly and was arrested later at the transition to the globular and the heart stage, respectively. These data demonstrate a strict dependence of cell division and embryo development on functional plastids as well as specific functions of plastids at different stages of embryogenesis. In addition, our analysis suggest that not all components of the translocase are equally essential for plastid protein import in vivo.     

Ionizing radiation and genetic risks. II. Nature of radiation-induced mutations in experimental mammalian in vivo systems

This paper reviews data on the nature of spontaneous and radiation-induced mutations in the mouse. The data are from studies using a variety of endpoints scorable at the morphological or the biochemical level and include pre-selected as well as unselected loci at which mutations can lead to recessive or dominant phenotypes. The loci used in the morphological recessive specific-locus tests permit the recovery of a wide spectrum of induced changes. Important variables that affect the nature of radiation-induced mutations (assessed primarily using tests for viability of homozygotes) include: germ cell stage, type of irradiation and the locus. Most of the results pertain to irradiated stem cell spermatogonia. The data on morphological specific-locus mutations show that overall, more than two-thirds of the X- or gamma-ray-induced mutations are lethal when homozygous. This proportion may be lower for those that occur spontaneously, but the numbers of tested mutants are small. For spontaneous mutations, there is evidence for the occurrence of mosaics and for proviral insertions. Most or all tested induced enzyme activity variants, dominant visibles (recovered in specific-locus experiments) and dominant skeletal mutations are lethal when homozygous and this is true of 50% of dominant cataract mutations, but again, the numbers of tested mutants are small. Electrophoretic mobility variants, which are known to be due to base-pair changes, are seldom induced by irradiation. At the histocompatibility loci, no radiation-induced mutations have been recovered, presumably because deletions are incompatible with survival even in heterozygotes. All these findings are consistent with the view that in mouse germ cells, most radiation-induced mutations are DNA deletions. Some mutations (in the morphological specific-locus tests) which had previously been inferred to be deletions on the basis of genetic analyses have now been shown to be DNA deletions by molecular methods. However, the possibility cannot be excluded that at least a small proportion of induced mutations may be intragenic changes. The data on the rates of induction of recessive lethals and of dominant skeletal and dominant cataract mutations (and proportions of the latter two which are homozygous lethal) can be used to estimate the proportions of recessive lethals which are expressed as skeletal abnormalities or cataracts. These calculations show that about 10% of recessive lethals manifest themselves as skeletal and less than 0.2% as cataract mutations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sequential steps for developmental arrest in Arabidopsis seeds

The continuous growth of the plant embryo is interrupted during the seed maturation processes which results in a dormant seed. The embryo continues development after germination when it grows into a seedling. The embryo growth phase starts after morphogenesis and ends when the embryo fills the seed sac. Very little is known about the processes regulating this phase. We describe mutants that affect embryo growth in two sequential developmental stages. Firstly, embryo growth arrest is regulated by the FUS3/LEC type genes, as mutations in these genes cause a continuation of growth in immature embryos. Secondly, a later stage of embryo dormancy is regulated by ABI3 and abscisic acid; abi3 and aba1 mutants exhibit premature germination only after embryos mature. Mutations affecting both developmental stages result in an additive phenotype and double mutants are highly viviparous. Embryo growth arrest is regulated by cell division activities in both the embryo and the endosperm, which are gradually switched off at the mature embryo stage. In the fus3/lec mutants, however, cell division in both the embryo and endosperm is not arrested, but rather is prolonged throughout seed maturation. Furthermore ectopic cell division occurs in seedlings. Our results indicate that seed dormancy is secured via at least two sequential developmental processes: embryo growth arrest, which is regulated by cell division and embryo dormancy.     

Maternal diabetes and oocyte quality

Maternal diabetes has been demonstrated to adversely affect preimplantation embryo development and pregnancy outcomes. Emerging evidence has implicated that these effects are associated with compromised oocyte competence. Several developmental defects during oocyte maturation in diabetic mice have been reported over past decades. Most recently, we further identified the structural, spatial and metabolic dysfunction of mitochondria in oocytes from diabetic mice, suggesting the impaired oocyte quality. These defects in the oocyte may be maternally transmitted to the embryo and then manifested later as developmental abnormalities in preimplantation embryo, congenital malformations, and even metabolic disease in the offspring. In this paper, we briefly review the effects of maternal diabetes on oocyte quality, with a particular emphasis on the mitochondrial dysfunction. The possible connection between dysfunctional oocyte mitochondria and reproductive failure of diabetic females, and the mechanism(s) by which maternal diabetes exerts its effects on the oocyte are also discussed.     

Monoclonal antibody probes discriminate early and late mutant defects in development of the Drosophila retina

Many mutations in Drosophila melanogaster affect the morphology of the adult compound eye. However, the times at which the phenotypes first become manifest in development are, in most cases, unknown; they can occur at any of a series of stages. Among mutants in which eyes appear externally similar, the developmental stage of onset of each defect may be quite different. Pattern formation in the compound eye begins during the late third larval instar in the eye imaginal disc, when a wave of morphogenesis crosses the disc from posterior to anterior. As this wave crosses the disc, there appears in its wake an array of photoreceptor neuron clusters and accessory cells that will comprise the adult ommatidia. Eye discs from 20 abnormal-eye mutants were analyzed using monoclonal antibodies that highlight various aspects of the developing array, to observe the stage at which each anomaly becomes evident. Some mutations apparently affect precursor cells, others the setting up of the pattern, others maintenance of the pattern, and still others later morphogenetic events.     

Late embryo-defective mutants of Arabidopsis

The embryo-defective (emb) mutants of Arabidopsis constitute a large and diverse group of mutants disrupted in a broad range of embryonic processes, including morphogonesis, cell differentiation, and maturation programs. This report describes a subset of these mutants, the late embryo defectives, which develop beyond the globular stage of embryogenesis but fail to complete normal morphogenesis. A representative sample of 12 late mutants was chosen for this study, patterns of morphogenesis were characterized, the germination potential of mutant seeds was investigated, and additional mutant alleles within the collection were identified. Morphological defects in mutant embryos became apparent during the heart stage of development, when embryos normally begin the rapid cell division and expansion required for the completion of morphogenesis. Despite their morphological abnormalities, mutant embryos often germinated from dry seed, demonstrating that genetic programs required for the establishment of desiccation tolerance remained intact. Mutant seedlings displayed a wide range of developmental abnormalities, including altered morphology, lack of pigmentation, dwarfism, and disorganized vegetative growth. One late mutant was found to be allelic to an early embryo defective that arrests at the globular stage. These results suggest that a number of late EMB genes encode basic cellular and metabolic functions needed for cell division, enlargement, and embryonic growth. The rapid growth and metabolic changes that occur at the heart stage may present a barrier to normal development in the late mutants, resulting in altered embryo morphology and other developmental defects. It is proposed that many Arabidopsis mutants with abnormal embryo and seedling morphology are not defective in the regulation of pattern formation or morphogenesis, but rather in fundamental physiological and cellular processes required for the completion of normal growth and development. © 1995 Wiley-Liss, Inc.     

L(1)trol and L(1)devl, Loci Affecting the Development of the Adult Central Nervous System in Drosophila Melanogaster

Adult optic lobes of Drosophila melanogaster are composed of neurons specific to the adult which develop postembryonically. The structure of the optic lobes and aspects of its development have been described, and a number of mutants that affect its development have been identified. The focus of every screen to date has been on disruption of adult structure or function. Although these loci were originally identified on the basis of viable mutants, some have proven capable of giving rise to lethal alleles. It seems reasonable to assume that mutants which strongly affect development of the imaginal-specific central nervous system may evidence abnormalities during the late larval or pupal stages when the adult central nervous system is undergoing final assembly and might show a lethal phase prior to eclosion (as is true for mutations at the previously defined l(1)ogre locus). We have carried out the first screen of autosomal and sex-linked late larval and pupal lethals to identify mutations that affect the development of the optic lobes. Our screen yielded nine mutants that could tentatively be grouped into three classes, depending on the neuroblast population affected and imaginal disc phenotypes. Two of these, including one that is allelic to l(1)zw1, were chosen for further analysis.     

Tubby-RFP balancers for developmental analysis: FM7c 2xTb-RFP, CyO 2xTb-RFP, and TM3 2xTb-RFP

We report here the construction of Tubby-RFP balancers for the X, 2nd and 3rd chromosomes of Drosophila melanogaster. The insertion of a 2xTb-RFP transgene on the FM7c, CyO, and TM3 balancer chromosomes introduces two easily scorable, dominant, developmental markers. The strong Tb phenotype is visible to the naked eye at the larval L2, L3, and pupal stages. The RFP associated with the cuticle is easily detected at all stages from late embryo to adult with the use of a fluorescence stereomicroscope. The FM7c Bar 2xTb-RFP, CyO Cy 2xTb-RFP, and TM3 Sb 2xTb-RFP balancers will greatly facilitate the analysis of lethals and other developmental mutants in L2/L3 larvae and pupae, but also provide coverage of other stages beginning in late embryogenesis through to the adult.     

Genome‐scale gene/reaction essentiality and synthetic lethality analysis

Synthetic lethals are to pairs of non‐essential genes whose simultaneous deletion prohibits growth. One can extend the concept of synthetic lethality by considering gene groups of increasing size where only the simultaneous elimination of all genes is lethal, whereas individual gene deletions are not. We developed optimization‐based procedures for the exhaustive and targeted enumeration of multi‐gene (and by extension multi‐reaction) lethals for genome‐scale metabolic models. Specifically, these approaches are applied to iAF1260, the latest model of Escherichia coli, leading to the complete identification of all double and triple gene and reaction synthetic lethals as well as the targeted identification of quadruples and some higher‐order ones. Graph representations of these synthetic lethals reveal a variety of motifs ranging from hub‐like to highly connected subgraphs providing a birds‐eye view of the avenues available for redirecting metabolism and uncovering complex patterns of gene utilization and interdependence. The procedure also enables the use of falsely predicted synthetic lethals for metabolic model curation. By analyzing the functional classifications of the genes involved in synthetic lethals, we reveal surprising connections within and across clusters of orthologous group functional classifications.