Maternal-effect mutations altering the anterior-posterior pattern of the Drosophila embryo

Summary Mutations in seven different maternal-effect loci on the second chromosome of Drosophila melanogaster all cause alterations in the anterior-posterior pattern of the embryo. Mutations in torso (tor) and trunk (trk) delete the anterior- and posterior-most structures of the embryo. At the same time they shift cellular fates which are normally found in the subterminal regions of the embryo towards the poles. Mutations in vasa (vas), valois (vls), staufen (stau) and tudor (tud) cause two embryonic defects. For one they result in absence of polar plasm, polar granules and pole cells in all eggs produced by mutant females. Secondly, embryos developing inside such eggs show deletions of abdominal segments. In addition, embryos derived from staufen mothers lack anterior head structures, embryos derived from valois mothers frequently fail to cellularize properly. Mutations in exuperantia (exu) cause deletions of anterior head structures, similar to torso, trunk and staufen. However in exu, these head structures are replaced by an inverted posterior end which comprises posterior midgut, proctodeal region, and often malpighian tubules.The effects of all mutations can be traced back to the beginning stages of gastrulation, indicating that the alterations in cellular fates have probably taken place by that time. Analysis of embryos derived from double mutant mothers suggests that these three phenotypic groups of mutants interfere with three different, independent pathways. All three pathways seem to act additively on the system which specifies anterior-posterior cellular fates within the egg.     

Mutational analysis of morphogenesis of the maize embryo

Morphogenesis of the maize embryo is controlled by many genes. A group of 51 embryo-specific (emb) mutations representing at least 45 independent mutation events and many different gene loci have been isolated from active Robertson's Mutator stocks. The authors have reported previously that the embryo phenotype of 27 of these mutations, characterized by examining mature embryos in fresh dissection. The maximal development capacity of the 24 emb mutations are reported here which have not been reported previously. All result in retarded embryos that are morphologically abnormal. Three of the mutants are blocked during the first phase of morphogenesis, the period in which the basal-apical asymmetry is established and the embryo is regionalized into suspensor and embryo proper. Nineteen mutants are blocked during the second phase, the period in which radial asymmetry appears, the embryonic axis is established at a different angle than the original basal-apical axis of the zygote and the vegetative organ primordia of the adult plant make their first appearance. Two mutants are blocked or altered during the third phase, the period in which vegetative structures are elaborated. Some of the mutants affected in the first phase of morphogenesis may have defective mechanisms for establishing basal-apical asymmetry, including possibly the asymmetric distribution of morphogenic determinants. Similarly, some of the mutants affected in the second phase may be altered in the mechanisms establishing radial asymmetry and the origin of the meristems. Mutations of the first type may act as early as the first cell division when the zygote undergoes a transverse division, while mutations of the second type are likely to act during the proembryo and transition stages. Both types include mutations affecting embryo pattern formation. Mutations affecting the third phase of morphogenesis may identify genes regulating reiterative morphogenic processes of vegetative plant development and events of embryo maturation. This group of 24 mutations is like that reported previously in representing genes that are crucial to embryo morphogenesis.     

Evidence that the rb Locus Alters the Starch Content of Developing Pea Embryos through an Effect on ADP Glucose Pyrophosphorylase

The aim of this work was to discover whether the rb locus of peas (Pisum sativum L.) affects seed starch content through action on an enzyme of starch synthesis in the developing embryo. The phenotypic effects of this locus are like those of the better characterised, unlinked r locus, which affects seed starch content through action on starch-branching enzyme. Embryos recessive at one or both of these loci (RRrbrb, rrRbRb, rrrbrb) have lower starch contents from an early stage of development than embryos dominant at these loci (RRRbRb). Maximum catalytic activities of enzymes of the pathway from sucrose to starch (sucrose synthase EC 2.4.1.13, UDP glucose pyrophosphorylase EC 2.7.7.9, ADP glucose pyrophosphorylase EC 2.7.7.27, ADP glucose-starch synthase EC 2.4.1.21, starch-branching enzyme EC 2.4.1.18) were compared in developing embryos of three lines of rbrb peas and four lines of RbRb peas. The only consistent difference between the two sorts of embryo was in the activity of ADP glucose pyrophosphorylase, which was at least tenfold lower in rbrb than in RbRb embryos. The activity in rbrb embryos was in most cases less than the estimated rate of starch synthesis of RRRbRb embryos. We conclude that the effect of the rb locus on the starch content of pea seeds is mediated through an alteration in the activity of ADP glucose pyrophosphorylase in the developing embryo.     

Mutation Analysis of Inhibitory Guanine Nucleotide Binding Protein Alpha (GNAI) Loci in Young and Familial Pituitary Adenomas

Pituitary adenomas are neoplasms of the anterior pituitary lobe and account for 15–20% of all intracranial tumors. Although most pituitary tumors are benign they can cause severe symptoms related to tumor size as well as hypopituitarism and/or hypersecretion of one or more pituitary hormones. Most pituitary adenomas are sporadic, but it has been estimated that 5% of patients have a familial background. Germline mutations of the tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP) predispose to hereditary pituitary neoplasia. Recently, it has been demonstrated that AIP mutations predispose to pituitary tumorigenesis through defective inhibitory GTP binding protein (Gα_i) signaling. This finding prompted us to examine whether germline loss-of-function mutations in inhibitory guanine nucleotide (GTP) binding protein alpha (GNAI) loci are involved in genetic predisposition of pituitary tumors. To our knowledge, this is the first time GNAI genes are sequenced in order to examine the occurrence of inactivating germline mutations. Thus far, only somatic gain-of-function hot-spot mutations have been studied in these loci. Here, we have analyzed the coding regions of GNAI1 _, GNAI2, and GNAI3 in a set of young sporadic somatotropinoma patients (n = 32; mean age of diagnosis 32 years) and familial index cases (n = 14), thus in patients with a disease phenotype similar to that observed in AIP mutation carriers. In addition, expression of Gα_i proteins was studied in human growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH)-secreting and non-functional pituitary tumors. No pathogenic germline mutations affecting the Gα_i proteins were detected. The result suggests that loss-of-function mutations of GNAI loci are rare or nonexistent in familial pituitary adenomas.     

Genetic analysis of a mutation on appressorium formation in Magnaporthe grisea

The rice blast fungus, Magnaporthe grisea, forms a dome-shaped and darkly pigmented infection structure, an appressorium, to penetrate its host. Differentiation and maturation of appressoria are critical steps for successful infection. A spontaneous developmental mutant (MG01) defective in appressorium formation was found in this fungus. The mutant did not form appressoria either on inductive hydrophobic surfaces or on rice leaves. The addition of cyclic AMP or 1,16-hexadecanediol was not effective in inducing appressorium formation in this mutant. This mutant did not cause lesions on rice when inoculated with conidial suspension by spraying or injecting into the leaf sheath. Genetic analysis of the mutant indicated that the phenotype is under single gene control, designated APP5. Crosses with previously described appressorium defective mutants (app1⁻ and app3⁻) of Magnaporthe grisea suggested that the mutations are at different loci. Bulked segregant analysis was employed to obtain DNA markers linked to the APP5 locus.     

Systematic Functional Analysis of Bicaudal-D Serine Phosphorylation and Intragenic Suppression of a Female Sterile Allele of BicD

Protein phosphorylation is involved in posttranslational control of essentially all biological processes. Using mass spectrometry, recent analyses of whole phosphoproteomes led to the identification of numerous new phosphorylation sites. However, the function of most of these sites remained unknown. We chose the Drosophila Bicaudal-D protein to estimate the importance of individual phosphorylation events. Being involved in different cellular processes, BicD is required for oocyte determination, for RNA transport during oogenesis and embryogenesis, and for photoreceptor nuclei migration in the developing eye. The numerous roles of BicD and the available evidence for functional importance of BicD phosphorylation led us to identify eight phosphorylation sites of BicD, and we tested a total of 14 identified and suspected phosphoserine residues for their functional importance in vivo in flies. Surprisingly, all these serines turned out to be dispensable for providing sufficient basal BicD activity for normal growth and development. However, in a genetically sensitized background where the BicD^A40V protein variant provides only partial activity, serine 103 substitutions are not neutral anymore, but show surprising differences. The S103D substitution completely inactivates the protein, whereas S103A behaves neutral, and the S103F substitution, isolated in a genetic screen, restores BicD^A40V function. Our results suggest that many BicD phosphorylation events may either be fortuitous or play a modulating function as shown for Ser¹⁰³. Remarkably, amongst the Drosophila serines we found phosphorylated, Ser¹⁰³ is the only one that is fully conserved in mammalian BicD.     

Drosophila gene tao-1 encodes proteins with and without a Ste20 kinase domain that affect cytoskeletal architecture and cell migration differently

Tao-1, the single representative of the Sterile 20 kinase subfamily in Drosophila, is best known for destabilizing microtubules at the actin-rich cortex, regulating the cytoskeletal architecture of cells. More recently, Tao-1 was shown to act in the Salvador–Warts–Hippo pathway by phosphorylating Hippo, regulating cell growth as well as cell polarity. Here, we show that tao-1 encodes two proteins, one with the Sterile 20 kinase domain (Tao-L) and one without it (Tao-S), and that they act in an antagonistic manner. Tao-L expression causes lamellipodia-like cell protrusions, whereas Tao-S expression results in filopodia-like structures that make cells stick to the surface they attach to. Ectopic Tao-1 expression in the anterior region of Drosophila embryos results in pole cell formation as normally observed at the posterior end. Tao-S expression causes primordial germ cells (PGCs) to adhere to the inner wall of the gut primordia and prevents proper transepithelial migration to the gonads. Conversely, RNAi knockdowns of Tao-1 cause disordered migration of PGCs out of the gut epithelium, their dispersal within the embryo and cell death. The results reveal a novel function of Tao-1 in cell migration, which is based on antagonistic activities of two proteins encoded by a single gene.     

Linkage Disequilibrium between Allozymes in Natural Populations of Lodgepole Pine

Pairwise linkage disequilibrium values (D) were estimated for 14 allozyme loci in two natural populations of lodgepole pine (Pinus contorta ssp. latifolia). Maternal multilocus genotypes were inferred from samples of (haploid) megagametophytic seed-endosperms. Coupling/repulsion double heterozygotes were distinguished for closely linked pairs of loci. Assays of seven of the loci in seed embryos allowed estimates of D for these loci in the outcross pollen pool (estimates of outcrossing rates indicate no significant departures from random mating in either population). No disequilibrium was observed between unlinked loci in either maternal genotypes or outcross pollen. However, significant disequilibrium was observed within and between gametes for some allelic combinations of four tightly linked loci; the assumption of random association of gamete types within individuals is thus invalid for some loci in lodgepole pine. Possible causes of the observed D were examined using the noncentrality parameter of the general noncentral chi square distribution. We concluded, from estimates of population size, linkage and measurements of population substructure, that neither drift nor population subdivision was responsible for the significant values of D which were observed and that epistatic selection was the most likely cause of the disequilibrium observed.     

Maternal control of embryogenesis by MPK6 and its upstream MKK4/MKK5 in Arabidopsis

In flowering plants, developing embryos reside in maternal sporophytes. It is known that maternal generation influences the development of next‐generation embryos; however, little is known about the signaling components in the process. Previously, we demonstrated that Arabidopsis mitogen‐activated protein kinase 6 (MPK6) and MPK3 play critical roles in plant reproduction. In addition, we noticed that a large fraction of seeds from mpk6 single‐mutant plants showed a wrinkled seed coat or a burst‐out embryo phenotype. Here, we report that these seed phenotypes can be traced back to defective embryogenesis. The defective embryos have shorter suspensors and reduced growth along the longitudinal axis. Furthermore, the cotyledons fail to bend over to progress to the bent‐cotyledon stage. As a result of the uneven circumference along the axis, the seed coat wrinkles to develop raisin‐like morphology after dehydration. In more severe cases, the embryo can be pushed out from the micropylar end, resulting in the burst‐out embryo seed phenotype. Genetic analyses demonstrated that the defective embryogenesis of the mpk6 mutant is a maternal effect. Heterozygous or homozygous mpk6 embryos have defects only in mpk6 homozygous maternal plants, but not in wild‐type or heterozygous maternal plants. The loss of function of MKK4/MKK5 also results in the same phenotypes, suggesting that MKK4/MKK5 might act upstream of MPK6 in this pathway. The maternal‐mediated embryo defects are associated with changes in auxin activity maxima and PIN localization. In summary, this research demonstrates that the Arabidopsis MKK4/MKK5–MPK6 cascade is an important player in the maternal control of embryogenesis.     

The Requirement of WHIRLY1 for Embryogenesis Is Dependent on Genetic Background in Maize

Plastid gene expression is essential to embryogenesis in higher plants, but the underlying mechanism is obscure. Through molecular characterization of an embryo defective 16 (emb16) locus, here we report that the requirement of plastid translation for embryogenesis is dependent on the genetic background in maize (Zea mays). The emb16 mutation arrests embryogenesis at transition stage and allows the endosperm to develop largely normally. Molecular cloning reveals that Emb16 encodes WHIRLY1 (WHY1), a DNA/RNA binding protein that is required for genome stability and ribosome formation in plastids. Interestingly, the previous why1 mutant alleles (why1-1 and why1-2) do not affect embryogenesis, only conditions albino seedlings. The emb16 allele of why1 mutation is in the W22 genetic background. Crosses between emb16 and why1-1 heterozygotes resulted in both defective embryos and albino seedlings in the F1 progeny. Introgression of the emb16 allele from W22 into A188, B73, Mo17, Oh51a and the why1-1 genetic backgrounds yielded both defective embryos and albino seedlings. Similar results were obtained with two other emb mutants (emb12 and emb14) that are impaired in plastid protein translation process. These results indicate that the requirement of plastid translation for embryogenesis is dependent on genetic backgrounds, implying a mechanism of embryo lethality suppression in maize.     

Uric Acid Accumulation in an Arabidopsis Urate Oxidase Mutant Impairs Seedling Establishment by Blocking Peroxisome Maintenance

Purine nucleotides can be fully catabolized by plants to recycle nutrients. We have isolated a urate oxidase (uox) mutant of Arabidopsis thaliana that accumulates uric acid in all tissues, especially in the developing embryo. The mutant displays a reduced germination rate and is unable to establish autotrophic growth due to severe inhibition of cotyledon development and nutrient mobilization from the lipid reserves in the cotyledons. The uox mutant phenotype is suppressed in a xanthine dehydrogenase (xdh) uox double mutant, demonstrating that the underlying cause is not the defective purine base catabolism, or the lack of UOX per se, but the elevated uric acid concentration in the embryo. Remarkably, xanthine accumulates to similar levels in the xdh mutant without toxicity. This is paralleled in humans, where hyperuricemia is associated with many diseases whereas xanthinuria is asymptomatic. Searching for the molecular cause of uric acid toxicity, we discovered a local defect of peroxisomes (glyoxysomes) mostly confined to the cotyledons of the mature embryos, which resulted in the accumulation of free fatty acids in dry seeds. The peroxisomal defect explains the developmental phenotypes of the uox mutant, drawing a novel link between uric acid and peroxisome function, which may be relevant beyond plants.     

Proximal-distal pattern formation in Drosophila: cell autonomous requirement for Distal-less gene activity in limb development

Limb development in the Drosophila embryo requires a pattern-forming system to organize positional information along the proximal–distal axis of the limb. This system must function in the context of the well characterized anterior–posterior and dorsal–ventral pattern-forming systems that are required to organize the body plan of the embryo. By genetic criteria the Distal-less gene appears to play a central role in limb development. Lack-of-function Distal-less mutations cause the deletion of a specific subset of embryonic peripheral sense organs that represent the evolutionary remnants of larval limbs. Distal-less activity is also required in the imaginal discs for the development of adult limbs. This requirement is cell autonomous and region specific within the developing limb primordium. Production of genetically mosaic imaginal discs, in which clones of cells lack Distal-less activity, indicates the existence of an organized proximal–distal positional information in very young imaginal disc primordia. We suggest that this graded positional information may depend on the activity of the Distal-less gene.     

Two ligands signal through the Drosophila PDGF/VEGF receptor to ensure proper salivary gland positioning

The Drosophila embryonic salivary gland is a migrating tissue that undergoes a stereotypic pattern of migration into the embryo. We demonstrate that the migratory path of the salivary gland requires the PDGF/VEGF pathway. The PDGF/VEGF receptor, Pvr, is strongly expressed in the salivary glands, and Pvr mutations cause abnormal ventral curving of the glands, suggesting that Pvr is involved in gland migration. Although the Pvr ligands, Pvf1 and Pvf2, have distinct expression patterns in the Drosophila embryo, mutations for either one of the ligands result in salivary gland migration defects similar to those seen in embryos that lack Pvr. Rescue experiments indicate that the PDGF/VEGF pathway functions autonomously in the salivary gland. The results of this study demonstrate that the Drosophila PDGF/VEGF pathway is essential for proper positioning of the salivary glands.     

Deubiquitylation Machinery Is Required for Embryonic Polarity in Caenorhabditis elegans

The Caenorhabditis elegans one-cell embryo polarizes in response to a cue from the paternally donated centrosome and asymmetrically segregates cell fate determinants that direct the developmental program of the worm. We have found that genes encoding putative deubiquitylating enzymes (DUBs) are required for polarization of one-cell embryos. Maternal loss of the proteins MATH-33 and USP-47 leads to variable inability to correctly establish and maintain asymmetry as defined by posterior and anterior polarity proteins PAR-2 and PAR-3. The first observable defect is variable positioning of the centrosome with respect to the cell cortex and the male pronucleus. The severity of the polarity defects correlates with distance of the centrosome from the cortex. Furthermore, polarity defects can be bypassed by mutations that bring the centrosome in close proximity to the cortex. In addition we find that polarity and centrosome positioning defects can be suppressed by compromising protein turnover. We propose that the DUB activity of MATH-33 and USP-47 stabilizes one or more proteins required for association of the centrosome with the cortex. Because these DUBs are homologous to two members of a group of DUBs that act in fission yeast polarity, we tested additional members of that family and found that another C. elegans DUB gene, usp-46, also contributes to polarity. Our finding that deubiquitylating enzymes required for polarity in Schizosaccharomyces pombe are also required in C. elegans raises the possibility that these DUBs act through an evolutionarily conserved mechanism to control cell polarity.     

Dynamics of maternal and paternal effects on embryo and seed development in wild radish (Raphanus sativus)

Background and Aims

Variability in embryo development can influence the rate of seed maturation and seed size, which may have an impact on offspring fitness. While it is expected that embryo development will be under maternal control, more controversial hypotheses suggest that the pollen donor and the embryo itself may influence development. These latter possibilities are, however, poorly studied. Characteristics of 10-d-old embryos and seeds of wild radish (Raphanus sativus) were examined to address: (a) the effects of maternal plant and pollen donor on development; (b) the effects of earlier reproductive events (pollen tube growth and fertilization) on embryos and seeds, and the influence of embryo size on mature seed mass; (c) the effect of water stress on embryos and seeds; (d) the effect of stress on correlations of embryo and seed characteristics with earlier and later reproductive events and stages; and (e) changes in maternal and paternal effects on embryo and seed characteristics during development.

Methods

Eight maternal plants (two each from four families) and four pollen donors were crossed and developing gynoecia were collected at 10 d post-pollination. Half of the maternal plants experienced water stress. Characteristics of embryos and seeds were summarized and also compared with earlier and later developmental stages.

Key Results

In addition to the expected effects of the maternal plants, all embryo characters differed among pollen donors. Paternal effects varied over time, suggesting that there are windows of opportunity for pollen donors to influence embryo development. Water-stress treatment altered embryo characteristics; embryos were smaller and less developed. In addition, correlations of embryo characteristics with earlier and later stages changed dramatically with water stress.

Conclusions

The expected maternal effects on embryo development were observed, but there was also evidence for an early paternal role. The relative effects of these controls may change over time. Thus, there may be times in development when selection on the maternal, paternal or embryo contributions to development are more and less likely.