Re-organisation of the cytoskeleton during developmental programmed cell death in Picea abies embryos

Cell and tissue patterning in plant embryo development is well documented. Moreover, it has recently been shown that successful embryogenesis is reliant on programmed cell death (PCD). The cytoskeleton governs cell morphogenesis. However, surprisingly little is known about the role of the cytoskeleton in plant embryogenesis and associated PCD. We have used the gymnosperm, Picea abies, somatic embryogenesis model system to address this question. Formation of the apical-basal embryonic pattern in P. abies proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass on one pole and the terminally differentiated suspensor cells on the other, separated by the embryonal tube cells. The organisation of microtubules and F-actin changes successively from the embryonal mass towards the distal end of the embryo suspensor. The microtubule arrays appear normal in the embryonal mass cells, but the microtubule network is partially disorganised in the embryonal tube cells and the microtubules disrupted in the suspensor cells. In the same embryos, the microtubule-associated protein, MAP-65, is bound only to organised microtubules. In contrast, in a developmentally arrested cell line, which is incapable of normal embryonic pattern formation, MAP-65 does not bind the cortical microtubules and we suggest that this is a criterion for proembryogenic masses (PEMs) to passage into early embryogeny. In embryos, the organisation of F-actin gradually changes from a fine network in the embryonal mass cells to thick cables in the suspensor cells in which the microtubule network is completely degraded. F-actin de-polymerisation drugs abolish normal embryonic pattern formation and associated PCD in the suspensor, strongly suggesting that the actin network is vital in this PCD pathway.     

Programmed cell death eliminates all but one embryo in a polyembryonic plant seed

Development of multiple embryos from a single zygote, the phenomenon called monozygotic polyembryony, is a widespread reproductive strategy found in higher plants and especially in gymnosperms. The enigma of plant monozygotic polyembryony is that only one embryo in a polyembryonic seed usually survives while the others are eliminated at an early stage. Here we report that programmed cell death (PCD) is the major mechanism responsible for elimination of subordinate embryos in a polyembryonic seed. Using post-fertilized pine (Pinus sylvestris) ovules, we show that once the dominant embryo is selected and, subsequently, the entire female gametophyte is affected by PCD, the cells of subordinate embryos initiate an autolytic self-destruction program. The progression of embryonic PCD follows a rigid basal-apical pattern, first killing the most basally situated cells, adjacent to the suspensor, and then proceeding towards the apical region until all cells in the embryonal mass are doomed. Our data demonstrate that during polyembryony, PCD serves to halt competition among monozygotic embryos in order to ensure survival of one embryo.     

Propagation of Norway spruce via somatic embryogenesis

Somatic embryogenesis combined with cryopreservation is an attractive method to propagate Norway spruce (Picea abies) vegetatively both as a tool in the breeding programme and for large-scale clonal propagation of elite material. Somatic embryos are also a valuable tool for studying regulation of embryo development. Embryogenic cell lines of Norway spruce are established from zygotic embryos. The cell lines proliferate as proembryogenic masses (PEMs). Somatic embryos develop from PEMs. PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos. Withdrawal of plant growth regulators (PGRs) stimulates PEM-to-somatic embryo transition accompanied by programmed cell death (PCD) in PEMs. This PCD is mediated by a marked decrease in extracellular pH. If the acidification is abolished by buffering the culture medium, PEM-to-somatic embryo transition together with PCD is inhibited. Cell death, induced by withdrawal of PGRs, can be suppressed by extra supply of lipo-chitooligosaccharides (LCOs). Extracellular chitinases are probably involved in production and degradation of LCOs. During early embryogeny, the embryos form an embryonal mass surrounded by a surface layer. The formation of a surface layer is accompanied by a switch in the expression pattern of an Ltp-like gene (Pa18) and a homeobox gene (PaHB1), from ubiquitous expression in PEMs to surface layer-specific in somatic embryos. Ectopic expression of Pa18 and PaHB1 leads to an early developmental block. Transgenic embryos and plants of Norway spruce are routinely produced by using a biolistic approach. The transgenic material is used for studying the importance of specific genes for regulating plant development, but transgenic plants can also be used for identification of candidate genes for use in the breeding programme.     

Inhibited polar auxin transport results in aberrant embryo development in Norway spruce

Current hypotheses concerning the role of polar auxin transport in embryo development are entirely based on studies of angiosperms, while little is known about how auxin regulates pattern formation in gymnosperms. In this study, different developmental stages of somatic embryos of Norway spruce (Picea abies) were treated with the polar auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Effects of the treatments on auxin content, embryo differentiation and programmed cell death (PCD) were analysed. During early embryo development, NPA-treatment led to increased indole-3-acetic acid (IAA) content, abnormal cell divisions and decreased PCD, resulting in aberrant development of embryonal tube cells and suspensors. Mature embryos that had been treated with NPA showed both apical and basal abnormalities. Typically the embryos had abnormal cotyledon formation and irregular cell divisions in the area of the root meristem. Our results show that polar auxin transport is essential for the correct patterning of both apical and basal parts of conifer embryos throughout the whole developmental process. Furthermore, the aberrant morhologies of NPA-treated spruce embryos are comparable with several auxin response and transport mutants in Arabidopsis. This suggests that the role of polar auxin transport is conserved between angiosperms and gymnosperms.     

Expression and regulation of genes associated with cell death during murine preimplantation embryo development

The newly fertilized preimplantation embryo depends entirely on maternal mRNAs and proteins deposited and stored in the oocyte prior to its ovulation. If the oocyte is not sufficiently equipped with maternally stored products, or if zygotic gene expression does not commence at the correct time, the embryo will die. One of the major abnormalities observed during early development is cellular fragmentation. We showed previously that cellular fragmentation in human embryos can be attributed to programmed cell death (PCD). Here, we demonstrate that the PCD that occurs during the 1-cell stage of mouse embryogenesis is likely to be regulated by many cell death genes either maternally inherited or transcribed from the embryonic genome. We have demonstrated for the first time the temporal expression patterns of nine cell death regulatory genes, and our preliminary experiments show that the expression of these genes is altered in embryos undergoing fragmentation. The expression of genes involved in cell death (MA-3, p53, Bad, and Bcl-xS) seems to be elevated, whereas the expression of genes involved in cell survival (Bcl-2) is reduced. We propose that PCD may occur by default in embryos that fail to execute essential developmental events during the first cell cycle. Mol. Reprod. Dev. 51:243–253, 1998. © 1998 Wiley-Liss, Inc.     

Bcl-2 rescues motoneurons from early cell death in the cervical spinal cord of the chicken embryo

Motoneurons (MNs) in the cervical spinal cord of the chicken embryo undergo programmed cell death (PCD) between embryonic day (E) 4 and E5. The intracellular molecules regulating this early phase of PCD remain unknown. Here we show that introduction of Bcl-2 by a replication-competent avian retroviral vector prevented MN degeneration at E4.5, whereas the expression of the green fluorescent protein (GFP) was ineffective. Bcl-2 expression did not affect the number of Islet-1/2-positive MNs at the onset of cell death (E4). However, when examined at the end of the cell death period (E5.5), the number of Islet-1/2-positive MNs was clearly increased in Bcl-2-transfected embryos compared with control and GFP-transfected embryos. Activation of caspase-3, which is normally observed in this early MN death, was also prevented by Bcl-2. Thus, MNs in the cervical spinal cord appear to use intracellular pathway(s) for early PCD that is responsive to Bcl-2.     

Loss of the maintenance methyltransferase, xDnmt1, induces apoptosis in Xenopus embryos

DNA methylation is necessary for normal embryogenesis in animals. Here we show that loss of the maintenance methyltransferase, xDnmt1p, triggers an apoptotic response during Xenopus development, which accounts for the loss of specific cell populations in hypomethylated embryos. Hypomethylation-induced apoptosis is accompanied by a stabilization in xp53 protein levels after the mid-blastula transition. Ectopic expression of HPV-E6, which promotes xp53 degradation, prevents cell death, implying that the apoptotic signal is mediated by xp53. In addition, inhibition of caspase activation by overexpression of Bcl-2 results in the development of cellular masses that resemble embryonic blastomas. Embryonic tissue explant experiments suggest that hypomethylation alters the developmental potential of early embryo cells and that apoptosis is triggered by differentiation. Our results imply that loss of DNA methylation in differentiated somatic cells provides a signal via p53 that activates cell death pathways.     

Many genomic regions are required for normal embryonic programmed cell death in Caenorhabditis elegans

To identify genes involved in programmed cell death (PCD) in Caenorhabditis elegans, we screened a comprehensive set of chromosomal deficiencies for alterations in the pattern of PCD throughout embryonic development. From a set of 58 deficiencies, which collectively remove approximately 74% of the genome, four distinct classes were identified. In class I (20 deficiencies), no significant deviation from wild type in the temporal pattern of cell corpses was observed, indicating that much of the genome does not contain zygotic genes that perform conspicuous roles in embryonic PCD. The class II deficiencies (16 deficiencies defining at least 11 distinct genomic regions) led to no or fewer-than-normal cell corpses. Some of these cause premature cell division arrest, probably explaining the diminution in cell corpse number; however, others have little effect on cell proliferation, indicating that the reduced cell corpse number is not a direct result of premature embryonic arrest. In class III (18 deficiencies defining at least 16 unique regions), an excess of cell corpses was observed. The developmental stage at which the extra corpses were observed varied among the class III deficiencies, suggesting the existence of genes that perform temporal-specific functions in PCD. The four deficiencies in class IV (defining at least three unique regions), showed unusually large corpses that were, in some cases, attributable to extremely premature arrest in cell division without a concomitant block in PCD. Deficiencies in this last class suggest that the cell death program does not require normal embryonic cell proliferation to be activated and suggest that while some genes required for cell division might also be required for cell death, others are not. Most of the regions identified by these deficiencies do not contain previously identified zygotic cell death genes. There are, therefore, a substantial number of as yet unidentified genes required for normal PCD in C. elegans.     

The mitochondrion--an organelle commonly involved in programmed cell death in Arabidopsis thaliana

Plant cells undergoing programmed cell death (PCD) at late stages typically show chromatin condensation and endonucleolytic cleavage prior to obvious membrane or organelle ultrastructural changes. To investigate possible early PCD-associated events, we used microscopic observations and flow cytometry to quantitate mitochondrial membrane potential (DeltaPsim) changes during PCD at the single cell and population levels using Arabidopsis protoplasts. A DeltaPsim loss was commonly induced early during plant PCD and was important for PCD execution, as evidenced by the concomitant reduction of the change in DeltaPsim and PCD by cyclosporin A, which inhibits mitochondrial permeability transition pores in animal cells. DeltaPsim loss occurred prior to nuclear morphological changes and was only associated with mitochondrial cytochrome c release (an apoptotic trigger in animals) in response to one of three PCD elicitors. Three different stimuli in wild type implicated DeltaPsim changes in PCD: ceramide, protoporphyrin IX, and the hypersensitive response elicitor AvrRpt2. Additionally, the behavior of the conditional ectopic cell death mutant accelerated cell death2 and ACD2-overproducing plants also implicated DeltaPsim alteration as key for PCD execution. Because ACD2 is largely a chloroplast component in mature plants, the observation that the cell death in acd2 mutants requires changes in mitochondrial functions implicates communication between chloroplasts and mitochondria in mediating PCD activation. We suggest that DeltaPsim loss is a common early marker in plant PCD, similar to what has been documented in animals. However, unlike in animal cells, in plant cells, mitochondrial cytochrome c release is not an obligatory step in PCD control.     

Physiological rationale for responsiveness of mouse embryonic stem cells to gp130 cytokines

Embryonic stem cells are established directly from the pluripotent epiblast of the preimplantation mouse embryo. Their derivation and propagation are dependent upon cytokine-stimulated activation of gp130 signal transduction. Embryonic stem cells maintain a close resemblance to epiblast in developmental potency and gene expression profile. The presumption of equivalence between embryonic stem cells and epiblast is challenged, however, by the finding that early embryogenesis can proceed in the absence of gp130. To explore this issue further, we have examined the capacity of gp130 mutant embryos to accommodate perturbation of normal developmental progression. Mouse embryos arrest at the late blastocyst stage when implantation is prevented. This process of diapause occurs naturally in lactating females or can be induced experimentally by removal of the ovaries. We report that gp130(-/-) embryos survive unimplanted in the uterus after ovariectomy but, in contrast to wild-type or heterozygous embryos, are subsequently unable to resume development. Inner cell masses explanted from gp130(-/-) delayed blastocysts produce only parietal endoderm, a derivative of the hypoblast. Intact mutant embryos show an absence of epiblast cells, and Hoechst staining and TUNEL analysis reveal a preceding increased incidence of cell death. These findings establish that gp130 signalling is essential for the prolonged maintenance of epiblast in vivo, which is commonly required of mouse embryos in the wild. We propose that the responsiveness of embryonic stem cells to gp130 signalling has its origin in this adaptive physiological function.     

Bcl‐2 rescues motoneurons from early cell death in the cervical spinal cord of the chicken embryo

Motoneurons (MNs) in the cervical spinal cord of the chicken embryo undergo programmed cell death (PCD) between embryonic day (E) 4 and E5. The intracellular molecules regulating this early phase of PCD remain unknown. Here we show that introduction of Bcl‐2 by a replication‐competent avian retroviral vector prevented MN degeneration at E4.5, whereas the expression of the green fluorescent protein (GFP) was ineffective. Bcl‐2 expression did not affect the number of Islet‐1/2‐positive MNs at the onset of cell death (E4). However, when examined at the end of the cell death period (E5.5), the number of Islet‐1/2‐positive MNs was clearly increased in Bcl‐2‐transfected embryos compared with control and GFP‐transfected embryos. Activation of caspase‐3, which is normally observed in this early MN death, was also prevented by Bcl‐2. Thus, MNs in the cervical spinal cord appear to use intracellular pathway(s) for early PCD that is responsive to Bcl‐2. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 381–390, 2002     

The Arabidopsis peptide kiss of death is an inducer of programmed cell death

Programmed cell death (PCD) has a key role in defence and development of all multicellular organisms. In plants, there is a large gap in our knowledge of the molecular machinery involved at the various stages of PCD, especially the early steps. Here, we identify kiss of death (KOD) encoding a 25-amino-acid peptide that activates a PCD pathway in Arabidopsis thaliana. Two mutant alleles of KOD exhibited a reduced PCD of the suspensor, a single file of cells that support embryo development, and a reduced PCD of root hairs after a 55°C heat shock. KOD expression was found to be inducible by biotic and abiotic stresses. Furthermore, KOD expression was sufficient to cause death in leaves or seedlings and to activate caspase-like activities. In addition, KOD-induced PCD required light in leaves and was repressed by the PCD-suppressor genes AtBax inhibitor 1 and p35. KOD expression resulted in depolarization of the mitochondrial membrane, placing KOD above mitochondria dysfunction, an early step in plant PCD. A KOD∷GFP fusion, however, localized in the cytosol of cells and not mitochondria.     

Programmed cell death in the ascidian embryo: modulation by FoxA5 and Manx and roles in the evolution of larval development

Programmed cell death (PCD) has been discounted in the ascidian embryo because the descendants of every embryonic cell appear to be present in the tadpole larva. Here we show that apoptotic PCD is initiated in the epidermis and central nervous system (CNS) but not in the endoderm, mesenchyme, muscle, and notochord cells during embryogenesis in molgulid ascidians. However, the affected cells do not actually die until the beginning of metamorphosis. Although specific patterns of PCD were different in distantly related ascidian species, the results suggest that removal of CNS cells by apoptosis is a urchordate feature predating the origin of the vertebrates. Certain molgulid ascidian species have evolved an anural (tailless) larva in which notochord cells fail to undergo the morphogenetic movements culminating in tail development. These anural species include Molgula occulta, the sister species of the urodele (tailed) species Molgula oculata. We show that PCD in the notochord cell lineage precedes the arrest of tail development in M. occulta and other independently evolved anural species. The notochord cells are rescued from PCD and a tail develops in hybrid embryos produced by fertilizing M. occulta eggs with M. oculata sperm, implying that apoptosis is controlled zygotically. Antisense inhibition experiments show that zygotic expression of the FoxA5 and Manx genes is required to prevent notochord PCD in urodele species and hybrids with restored tails. The results provide the first indication of PCD in the ascidian embryo and suggest that apoptosis modulated by FoxA5 and Manx is involved in notochord and tail regression during anural development. Differences in PCD that occur between ascidian species suggest that diversity in programming apoptosis may explain differences in larval form.     

A key developmental switch during Norway spruce somatic embryogenesis is induced by withdrawal of growth regulators and is associated with cell death and extracellular acidification.

The biotechnology of somatic embryogenesis holds considerable promise for clonal propagation and breeding programs in forestry. To efficiently regulate the whole process of plant regeneration through somatic embryogenesis, it is of outmost importance to understand early developmental events when somatic embryos are just formed. In Norway spruce, somatic embryos transdifferentiate from proembryogenic masses (PEMs). This work describes the developmental dynamics (frequency distribution of PEMs and early somatic embryos) of the whole embryogenic suspension culture growing in the presence and absence of plant growth regulators (PGRs), auxin and cytokinin. The experiments have shown that PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos and ultimately plant production. This switch was induced by the withdrawal of PGRs in cell suspension leading to a rapid accumulation of early somatic embryos (to a maximum of 75% of the entire population of suspension culture) and concomitant degradation of PEMs. The latter was evident from increased level of cell death measured through spectrophotometric Evans blue staining assay. Proembryogenic mass-to-embryo transition and concomitant activation of cell death were mediated by strong extracellular acidification. Therefore, buffering PGR-free culture medium at high (pH 5.8) or low (pH 4.5) levels of pH inhibited both PEM-to-embryo transition and cell death. The yield of mature somatic embryos on abscisic acid (ABA)-containing medium was increased up to 10-fold if the suspension culture had been pretreated for 1 to 9 days in unbuffered PGR-free medium. In this case a large proportion (75%) of the total number of mature embryos was formed within a short, 5-week, contact with ABA. The latter is practically important because prolonged contact with ABA suppresses the growth of somatic embryo plants. Based on these results, an improved method for regulating somatic embryogenesis was set up and tested for nine genotypes of Norway spruce. Over 800 plants regenerated from all tested genotypes demonstrated a good performance in the greenhouse and they were transferred to the field.     

小鼠胚胎干细胞分化形成拟胚体过程中的细胞程序性死亡

为了检测小鼠胚胎干细胞 (embryonicstemcell ,ES细胞 )体外分化的拟胚体 (embryoidbodies ,EBs)形成过程中细胞程序性死亡 (programmedcelldeath ,PCD)的发生 ,通过悬滴、悬浮培养技术定向诱导未分化的ES细胞分化为拟胚体 ,并用RT PCR检测原始内胚层、原始外胚层、中胚层、内脏内胚层 4种分子标记物在EBs中的表达 .通过TUNEL染色、电镜、激光共聚焦显微镜及Western印迹以确定凋亡发生 .结果表明 :ES细胞体外分化为拟胚体并且表达各胚层相应的分子标记物 ;在拟胚体的发育过程中出现明显的空腔化过程 ,TUNEL染色及电镜观察到凋亡生成 ,同时线粒体膜电位 (ΔΨm)在拟胚体发育过程中降低 ,通过Western印迹检测到caspase3、caspase8的激活 .表明小鼠ES细胞所分化的拟胚体可以作为研究早期胚胎发育的实验模型 ,线粒体在拟胚体的细胞程序性死亡过程中发挥重要的作用 .为进一步利用拟胚体研究细胞程序性死亡及相关信号分子在小鼠胚胎发育早期的作用奠定了基础     

DspA/E, a type III effector of Erwinia amylovora, is required for early rapid growth in Nicotiana benthamiana and causes NbSGT1-dependent cell death

DspA/E is a pathogenicity factor of Erwinia amylovora that is translocated into the plant cell cytoplasm through an Hrp type III secretion system. Transient expression of dspA/E in Nicotiana benthamiana or yeast induced cell death, as it does in N. tabacum and apple as described previously. DspA/E-induced cell death in N. benthamiana was not inhibited by coexpression of AvrPtoB of Pseudomonas syringae pv. tomato , which inhibits programmed cell death (PCD) induced by several other elicitors in plants. Silencing of NbSGT1 , the expression of which is required for PCD mediated by several resistance proteins of plants, prevented DspA/E-induced cell death in N. benthamiana. However, silencing of NbRAR1 , or two MAP kinase kinase genes, which are required for PCD associated with many resistance genes in plants, did not prevent cell death induced by DspA/E. Silencing of NbSGT1 also compromised non-host resistance against E. amylovora . E. amylovora grew rapidly within the first 24 h after infiltration in N. benthamiana , and DspA/E was required for this early rapid growth. However, bacterial cell numbers decreased after 24 h in TRV-vector-transformed plants, whereas a dspA/E mutant strain grew to high populations in NbSGT1 -silenced plants. Our results indicate that DspA/E enhances virulence of E. amylovora in N. benthamiana, but the bacteria are then recognized by the plant, resulting in PCD and death of bacterial cells or restriction of bacterial cell growth.     

Developmentally regulated expression of cell surface carbohydrates during mouse embryogenesis

Cell surface carbohydrates undergo marked alterations during mouse embryogenesis. In preimplantation embryos, many carbohydrate markers show stage-specific expression in diverse ways. In early postimplantation embryos, certain carbohydrate markers are localized in defined regions in the embryo. Important carriers of stage-specific carbohydrates are the lactoseries structure (Gal beta 1----4GlcNAc) and the globoseries structure (Gal alpha 1----4Gal). Notably, the glycoprotein-bound large carbohydrate of poly-N-acetyllactosamine-type ([Gal beta 1----4GlcNAc beta 1----3]n) carries a number of markers preferentially expressed in early embryonic cells. These markers are of practical value in analyzing embryogenesis and cell differentiation. For example, in order to monitor in vitro differentiation of multipotential embryonal carcinoma cells, stage-specific embryonic antigen-1 (SSEA-1) and the Lotus agglutinin receptor have been used as markers of the undifferentiated cells, and the Dolichos agglutinin receptor has been used as a marker of extraembryonic endoderm cells. Developmental control of cell surface carbohydrates is attained by controlled expression of activities of key glycosyltransferases; for example, the activity of N-acetylglucosaminide alpha 1----3 fucosyltransferase is lost during in vitro differentiation of embryonal carcinoma cells to parietal endoderm cells, in parallel to the disappearance of SSEA-1. Accumulating evidence suggests that poly-N-acetyllactosamine-type glycans that are abundant in early embryonic cells are involved in cell surface recognition of these cells.     

Polar auxin transport controls suspensor fate

Polar auxin transport is critical for normal embryo development in angiosperms. It has been proposed that auxin accumulates dynamically at specific positions, which in early Arabidopsis embryos correlates with developmental decisions such as specification of the apical cell lineage, specification of the hypophysis, and differentiation of the two cotyledons. In conifers, pattern formation during embryo development is different, and includes a free nuclear stage, nondividing suspensor cells, presence of tube cells, lack of hypophysis and formation of a crown of cotyledons surrounding the shoot apical meristem. We have recently shown that polar auxin transport is important for normal embryo development also in conifers. Here we suggest a model where auxin is transported from the suspensor cells to the embryonal mass during early embryogeny in conifers. This transport is essential for the developmental decisions of the tube cells and the suspensor, and affects both the amount of programmed cell death and the embryo patterning.Key words: conifer, embryo development, 1-N-naphtylphthalamic acid (NPA), patterning, polar auxin transport, programmed cell death, somatic embryogenesis, suspensorIn the model plant Arabidopsis thaliana auxin is transported, already from the first cell division of the zygote, from the basal cell to the apical cell, where it is involved in establishing the identity of the apical cell lineage. At the 32-cell stage the polar auxin transport is reversed, leading to an auxin accumulation in the uppermost suspensor cell, which occurs concomitantly with the specification of the hypophysis. During the heart stage auxin is transported towards the cotyledonary primordia, giving positional information about the cotyledon outgrowth.¹ Formation of the apical-basal embryonic pattern during early embryogeny in conifers is quite different from that in Arabidopsis and proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass, the embryonal tube cells and terminally differentiated nondividing suspensor cells.²The somatic embryo system of Picea abies (Norway spruce) includes a stereotyped sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments.^3,4 We are using this as a model system for elucidating the regulation of embryo development in conifers.² Early somatic embryos differentiate from proembryonic masses (PEMs) after withdrawal of the plant growth regulators (PGRs) auxin and cytokinin (Fig. 1A and B). We have previously shown that the organisation of the apical-basal polarity in early embryos is dependent on a gradient of PCD from the embryonal tube cells committed to death, to the cell corpses at the basal end of the suspensor.^5–7 Dysregulation of the PCD leads to aberrant apical-basal patterning.Open in a separate windowFigure 1Model for polar auxin transport control of early embryo patterning in conifers. (A) Embryogenic cultures proliferate as proembryonic masses (PEMs) in the presence of the plant growth regulators (PGRs) auxin and cytokinin. (B) Early embryos start to differentiate from PEMs after withdrawal of PGRs. Endogenous auxin is transported to the newly formed embryonal mass. (C) Early embryos are formed within two weeks in PGR-free medium. Early embryos have a distinct embryonal mass, tube cells and a suspensor. IAA is transported from the suspensor and the tube cells to the embryonal mass. (D) Fully matured cotyledonary embryos are formed after 5–6 weeks on maturation medium. (E) Treatment with NPA blocks the polar auxin transport to the embryonal mass, leading to an IAA accumulation in the suspensor cells, tube cells and perhaps also in the cells of the embryonal mass most adjacent to the tube cells. (F) Embryos with supernumerary suspensor cells are formed if polar auxin transport is inhibited only during the earliest stages of suspensor differentiation. (G) Embryos with meristematic cells in the suspensor are formed if polar auxin transport is inhibited during both differentiation and elongation of the suspensor. We assume that these abnormalities abort further development and maturation of viable embryos. em, embryonal mass; s, suspensor; tc, tube cells. Green arrows indicate polar auxin transport, T indicates blocked polar auxin transport, green shadings indicate auxin accumulation.We recently showed that in embryogenic cultures of Norway spruce treated with the polar auxin transport inhibitor NPA, the number of cells undergoing PCD decreases. As a consequence the balance between the number of cells in the embryonal mass and the number of cells in the suspensor develop abnormally, and concomitantly the endogenous free IAA content increases almost two-fold.⁸In order to visualise the IAA accumulation within the embryos we used a -318 bp deletion of the auxin-responsive IAA4/5 promoter from Pisum sativum (pea), previously characterized by Oeller et al.,⁹ and Ballas et al.,¹⁰ fused to the GUS reporter gene.¹¹ In tobacco (Nicotiana tabacum) the promoter is expressed in rapidly elongating hypocotyls,¹² (our unpublished observations) and strong induction by auxin is clear in elongating zones of both roots and hypocotyls in transgenic pIAA4/5-GUS Arabidopsis plants.¹¹ However, to our knowledge, expression of IAA4/5 has not been reported in embryonal shoot apical meristems. Hence, the pIAA4/5-GUS may preferentially be used as a biosensor of auxin activity in non-meristematic cells during spruce embryo development. During normal somatic embryo development in spruce, pIAA4/5-GUS activity is detected in PEMs, tube cells and suspensor cells, but not in the embryonal mass. Early embryos of Norway spruce that are treated with NPA show increased pIAA4/5-GUS activity in tube cells and suspensor cells (unpublished), well in line with the increment of free IAA levels.Our results indicate that IAA under normal conditions is transported from the suspensor cells to the cells in the embryonal mass (Fig. 1B and C). NPA-treatment blocks this polar transport of endogenous IAA, which results in an accumulation of IAA and increased pIAA4/5-GUS activity in the suspensor cells, the tube cells, and perhaps also in the cells of the embryonal mass most adjacent to the tube cells (Fig. 1F and G). Blocked polar auxin transport during early differentiation of the suspensor stimulates abnormal cell divisions of the meristematic cells most adjacent to the tube cells or perhaps even of the tube cells themselves. Consequently, embryos with supernumerary tube and suspensor cells are formed (Fig. 1F). If the polar auxin transport is blocked for a longer time, i.e., during both differentiation and elongation of the suspensor, the auxin accumulation leads to maintenance of meristematic fate and a failure to undergo PCD (Fig. 1G).It has been proposed that the fate of the suspensor cells is regulated by signals from the embryo proper which impede developmental potential and initiate PCD.¹³ In accordance, we assume that the abnormal embryo morphologies formed after NPA-treatment may result from adverse inhibitory signals from the embryonal mass.     

grim promotes programmed cell death of Drosophila microchaete glial cells

The Inhibitor of apoptosis (IAP) antagonists Reaper (Rpr), Grim and Hid are central regulators of developmental apoptosis in Drosophila. Ectopic expression of each is sufficient to trigger apoptosis, and hid and rpr have been shown to be important for programmed cell death (PCD). To investigate the role for grim in PCD, a grim null mutant was generated. grim was not a key proapoptotic gene for embryonic PCD, confirming that grim cooperates with rpr and hid in embryogenesis. In contrast, PCD of glial cells in the microchaete lineage required grim, identifying a death process dependent upon endogenous grim. Grim associates with mitochondria and has been shown to activate a mitochondrial death pathway distinct from IAP antagonization; therefore, the Drosophila bcl-2 genes buffy and debcl were investigated for genetic interaction with grim. Loss of buffy led to microchaete glial cell survival and suppressed death in the eye induced by ectopic Grim. This is the first example of a developmental PCD process influenced by buffy, and places buffy in a proapoptotic role. PCD of microchaete glial cells represents an exceptional opportunity to study the mitochondrial proapoptotic process induced by Grim.     

Programmed cell death in the embryonic central nervous system of Drosophila melanogaster

Although programmed cell death (PCD) plays a crucial role throughout Drosophila CNS development, its pattern and incidence remain largely uninvestigated. We provide here a detailed analysis of the occurrence of PCD in the embryonic ventral nerve cord (VNC). We traced the spatio-temporal pattern of PCD and compared the appearance of, and total cell numbers in, thoracic and abdominal neuromeres of wild-type and PCD-deficient H99 mutant embryos. Furthermore, we have examined the clonal origin and fate of superfluous cells in H99 mutants by DiI labeling almost all neuroblasts, with special attention to segment-specific differences within the individually identified neuroblast lineages. Our data reveal that although PCD-deficient mutants appear morphologically well-structured, there is significant hyperplasia in the VNC. The majority of neuroblast lineages comprise superfluous cells, and a specific set of these lineages shows segment-specific characteristics. The superfluous cells can be specified as neurons with extended wild-type-like or abnormal axonal projections, but not as glia. The lineage data also provide indications towards the identities of neuroblasts that normally die in the late embryo and of those that become postembryonic and resume proliferation in the larva. Using cell-specific markers we were able to precisely identify some of the progeny cells, including the GW neuron, the U motoneurons and one of the RP motoneurons, all of which undergo segment-specific cell death. The data obtained in this analysis form the basis for further investigations into the mechanisms involved in the regulation of PCD and its role in segmental patterning in the embryonic CNS.