In vitro fertilization and expression of transgenes in gametes and zygotes

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Abstract The in vitro fertilization system of maize isthe well characterized model system for the fertilizationprocess and early zygotic embryogenesis of higherplants. Application of molecular methods to the in vitrofertilization system led to the isolation of new genes anduncovered specific expression patterns of cell cycleregulators. Recent studies showed that expression oftransgenes is possible in gametes and zygotes, thus trans-genic approaches might offer an opportunity to unravelthe roles of genes during fertilization and early develop-ment. The competence of gametes and zygotes to expresstransgenes will also enable the expression of GFP basedreporter genes for the visualization of subcellular compo-nents in these cells in vivo. This review focuses on thedata concerning the expression of transgenes in gametesand zygotes and describes some examples of recentdevelopments in transgenic technology illustratingthe emerging possibilities in experimental design bycombining this technology with in vitro fertilization.Keywords In vitro fertilization, egg cell, central cell,zygote, transgene expressionIntroductionFertilization is the central event in the life cycle of higherorganisms. In higher plants two fertilization processesoccur (Nawaschin 1898): one sperm cell of a pollengrain fuses with the egg cell forming the zygote whichdevelops into the embryo, whereas the second sperm cellfuses with the central cell forming the primary endospermcell which develops into the endosperm. These events take place deeply embedded in maternaltissue. Therefore the isolation of gametes is necessary toperform fertilization in vitro. The fusion of isolatedgametes in angiosperms in vitro can be mediated electri-cally and chemically by calcium or polyethylene glycol(recently reviewed by Kranz and Kumlehn 1999). Inmaize, both fertilization processes are possible in vitroby electrofusion of isolated gametes and central cells.These in vitro fertilized egg and central cells are able todevelop in culture and are capable of self-organization ina typical manner independently from maternal tissue(Kranz et al. 1991; Kranz et al. 1998).Besides leading to new insights, cytological studiesshow that development of the fusion products in cultureis characteristic and comparable with the situation inplanta. In vitro produced zygotes are highly metabolicallyactive. Synthesis of a new cell wall starts as early as 30 safter fusion of the gametes (Kranz et al. 1995). Fusion ofthe two parental nuclei can take place 35 min afterfusion (Tirlapur et al. 1995), but generally karyogamyoccurs between 45 and 120 min after in vitro fertilizationof egg cells (Faure et al. 1993; Kranz et al. 1998). Twonucleoli were observed in zygotes 18 h after in vitrofertilization (Kranz et al. 1995). In planta maize zygotesare dividing about 16 h after karyogamy (Mol et al.1994). In vitro produced zygotes displayed an unequalfirst cell division as in planta, depending on the cultureconditions between 29 and 46 h after fertilization, andthey could be regenerated to phenotypically normal andfertile plants (Kranz and Lrz 1993). Karyogamy of invitro fertilized central cells takes place within 120 minafter fusion. The in vitro produced primary endospermcells develop into oblong structures. Early nucleardivisions are not followed by cell wall formation, conse-quently generating a syncytium. Cellularization occurs 3to 5 days after in vitro fertilization and further develop-ment leads to formation of a white compact tissue. Thedevelopment of in vitro produced endosperm is concludedas in planta (Kranz et al. 1998).In vitro fertilization systems provide the basis formolecular studies of the fertilization process, gametes,zygotes and early developmental stages without theinfluence of maternal tissue. To explore gene expressionduring fertilization and early development, cDNA librariesof egg cells and zygotes were generated (Dresselhaus etS. Scholten E. Kranz ()Institut fr Allgemeine Botanik, AMPII, Universitt Hamburg,Ohnhorststrae 18, 22609 Hamburg, Germanye-mail: ekranz@botanik.uni-hamburg.deSex Plant Reprod (2001) 14:3540 Springer-Verlag 2001R E V I E WStefan Scholten Erhard KranzIn vitro fertilization and expression of transgenesin gametes and zygotesReceived: 20 December 2001 / Revision accepted: 6 June 2001al. 1994, 1996). Differential analysis of these librariesshowed that expression of several genes is up- or down-regulated after fertilization (Dresselhaus et al. 1999).Screening these libraries and a cDNA library of maturepollen for clones encoding transcription factors led to theisolation of novel cDNAs encoding MADS box proteins(Heuer 1999; Heuer et al. 2000). Transgenic approachesdemonstrate the homeotic function of MADS box genesduring flower development (reviewed by Riechmann andMeyerowitz 1997). The presence of transcripts of two ofthe novel MADS box genes in egg cells (Heuer 1999)implicate a role of these genes during fertilization and/orzygote development.RT-PCR allows expression analysis of known genes.Applying this method, adapted to the analysis of singlecells by Richert et al. (1996), to the in vitro fertilizationsystem, Sauter et al. (1998) showed the differentialexpression pattern of cell cycle regulatory genes duringfertilization and the first embryonic cell cycle. Incontrast to the cdc2 protein kinase, which is constitutivelyexpressed before and after fertilization, cyclin genes aretranscribed de novo after fertilization and display adiverse expression pattern during fertilization and zygotedevelopment.As discussed, in vitro fertilization systems led to theisolation of new genes and revealed particular expressionpatterns of cell cycle regulators, both of which mighthave important roles during fertilization and zygotedevelopment. Studies on functions of these genes inearly developmental stages of the plant life cycle are ofhigh interest.Transgene technology provides a way to gain insightsinto gene function by altering the expression level of agiven gene with overexpression or expression of antisenseRNA. Many studies show that these techniques are aswell suited to uncover the role of genes important fordevelopment, like transcription factors, as to raise under-standing of the regulation of developmental processes(Ramachandran et al. 1994; Meisel and Lam 1997).Moreover, the novel marker green fluorescent protein(GFP), isolated from Aequorea victoria, extends thepossibilities of transgenic technology. Due to its non-toxicnature and the non-invasive visualization by fluores-cence microscopy GFP permits real-time observations ofdynamic changes in living cells. GFP fusion proteins canbe used to study subcellular localization and movementsof proteins and organelles in vivo (Grebenok et al. 1997;Khler 1998). Other GFP-based approaches, of specialinterest for the application to the in vitro fertilizationsystem, enable visualization of cytoskeleton proteins ormeasurement of intracellular calcium concentrations. Amicrotubule reporter gene (gfp-mbd) was constructed byfusing the gfp gene to the microtubule binding domainof the mammalian microtubule-associated protein 4(MAP4) gene. GFP-MBD labels cortical microtubulesafter transient expression of the reporter gene in livingepidermal cells of fava bean (Marc et al. 1998). Grangerand Cyr (2000) showed that constitutive expressionof the microtubule reporter gene in stable transformedtobacco BY-2 cells allows spatial and temporal resolutionof microtubule arrays as they reorganize throughout thecell cycle. By using GFP fusion proteins, which bind toactin, the visualization of dynamic changes of thiscomponent of the cytoskeleton could be achieved (Kostet al. 1998).Calcium ions play a central role in the regulation ofmetabolic processes and signal transduction (reviewedby Zhang and Cass 1997) and this ion might have a rolein egg activation (Antoine et al. 2000 this volume).Digonnet et al. (1997) reported an increase in Ca2+concentration in egg cells after fertilization. An influx ofextracellular Ca2+ induced by gamete fusion was measuredwith the use of an extracellular Ca2+ selective vibratingprobe by Antoine et al. (2000). Starting in the vicinity ofthe sperm cell fusion side, the Ca2+ influx spread sub-sequently through the whole egg cell plasma membraneas a wavefront. The GFP based cameleon calciumindicator, developed by Miyawaki et al. (1997) couldpossibly be used to further characterize the spatial andtemporal distribution of calcium ions during fertilizationand early embryonic development in vivo. Recently, thefunction of this indicator was shown in guard cells of Arabidopsis (Allen et al. 1999).These examples show that expression of transgenes inisolated gametes and zygotes produced in vitro willbecome a valuable tool for analysis of these develop-mental stages, for both functional analysis and cytologicalstudies.So far, there are two strategies to study expression offoreign genes in gametes and zygotes. One is directdelivery of DNA into these cells via microinjection.With the second method transgene expression in gametesand zygotes can be analyzed after regeneration of plantstransformed via particle bombardment.MicroinjectionTransient expression of transgenes after microinjectionof plasmid DNA in zygotes was reported by Leduc et al.(1996). In this study the GUS gene under control of themaize histone H3C4 promoter followed by an actinintron and two anthocyanin regulatory genes under controlof the 35S promoter were used as reporter genes. Thesevectors were injected in zygotes of maize that wereisolated 24 h after pollination. Transient expression ofthe reporter genes, with a frequency of 3.5% on average,was reported in zygotes 4 days after injection. Pnya etal. (1999) demonstrated transient expression of reportergenes after microinjection of plasmid DNA into egg cellsand isolated zygotes of wheat. A GFP gene under controlof the ubiquitin promoter was injected into egg cells,whereas the GUS gene driven by the 35S promoter wasinjected into zygotes. Transient expression frequenciesof 46% and 52% on average for egg cells and zygotes,respectively, could be achieved in these experiments.The authors suggested that high-frequency AC fields,applied to immobilize the cells on an electrode, might be36a reason for such high expression frequencies but thisremains to be determined.In general, immobilization of cells for microinjectionis performed with a holding capillary or by embeddingthe cells in low melting point agarose. After injection ofembedded, isolated maize zygotes we obtained transientexpression frequencies up to 30% (E. Kranz, unpublishedresults). In these experiments the gfp gene under controlof an enhanced 35S promoter followed by the first intronof the hsp70 gene (Pang et al. 1996) was used. GFPfluorescence was monitored about 18 h after injectionand culture (Fig. 1A). These studies focus on the transient expression oftransgenes after microinjection of plasmids into egg cellsor zygotes. This methods advantage is that results canbe obtained promptly after injection of DNA into a cellof interest. Therefore it could be a suitable method forevaluation of promoter activities in the target cells.Holm et al. (2000) demonstrated stable transformationof barley by microinjection of DNA into isolatedzygotes. The efficient regeneration system for isolatedbarley zygotes was the basis of these experiments. In thisco-culture system with barley microspores undergoingembryogenesis, the isolated zygotes developed intoembryo-like structures with a frequency of 75%. Fertileplants were regenerated from approximately 50% of theembryo-like structures (Holm et al. 1994). After micro-injection of the GUS gene under control of the rice actinpromoter into isolated barley zygotes, presence of theconstruct was confirmed by PCR with a mean frequencyof 21% of the derived structures. GUS expression wasfound in few cases. Two lines of green plants wereshown to be transgenic, one of them for an intact copy ofthe expression cassette beside fragments of the construct,but the GUS gene was not expressed. Degradation of theintroduced DNA was discussed as a possible reason forthe rarely found expression of the transgene after micro-injection (Holm et al. 2000).After circumvention of these problems, stable trans-formation by microinjection of zygotes would be of greatadvantage for applied purposes, since the use of selectablemarker genes is not required. The regenerants can bescreened directly for the presence of the transgene. Efficientregeneration systems for isolated zygotes, which are thebasis for this transformation method, were established forwheat (Kumlehn et al. 1997, 1998) and maize (Leduc etal. 1996). Also, maize zygotes produced in vitro can beefficiently regenerated into plants (Kranz and Lrz 1993).In vitro fertilization provides the possibility of injectingDNA into egg cells before fertilization. This option mighthave an impact on the integration event.Microprojectile bombardmentFor investigation of stably integrated transgene expressionin gametes and zygotes we generated transgenic plantsby microprojectile bombardment of immature embryos,following the protocol of Brettschneider et al. (1997).With this protocol transformation frequencies up to 4%were obtained. An advantage of stable transformationover transient expression assays is that transgenic linesestablished and characterized once can be used forvarious experiments without the need for new time-consuming microinjection experiments. Transgenes under control of constitutive promotersTo date there is little information about the regulationand activity of transcription and translation in gametesand zygotes. The competence of the female gametes forsynthesizing proteins quickly and in large amounts forfertilization-induced development is indicated by therelatively large number of ribosomes in egg cells andcentral cells of maize (Dow and Mascarenhas 1991) andthe high concentrations of poly(A)RNA, found in Capsellaegg cells (Raghavan 1990). This view is supported bythe presence of various specific transcripts found in eggcells and central cells of maize and the early expressionof cycline genes in maize zygotes (Sauter et al. 1998;Dresselhaus et al. 1999). Male gametes synthesize RNAand proteins. This synthesis was found in isolated malegametes of maize (Zhang et al. 1993), and transcriptionalactivity was shown for isolated nuclei of maize spermcells (Matthys-Rochon et al. 1994). There is evidence formale gametic cell-specific gene expression and proteinsynthesis in Lilium (Blomstedt et al. 1996; Xu et al.1999a,b). However, the onset of transcription of severalpaternally inherited alleles in the Arabidopsis embryowas reported to occur late after fertilization, in embryoswith 3264 cells (Vielle-Calzada et al. 2000).To gain insight into the competence of maize gametesand zygotes for expressing stable integrated transgenes,transgenic plants with the GFP vector constructed byPang et al. (1996) were generated. This vector was usedbecause it is optimized for high expression levels of GFPin monocotyl plants and codes for a plant codon usage-optimized S65T version of the GFP.37Fig. 1A, B GFP expression in maize zygotes and cells of theembryo sac. The 35S:gfp construct (Pang et al. 1996) was used.A GFP fluorescence in zygotes 18 h after injection of DNA andculture. B GFP fluorescence of a stable transgenic maize plant.Isolated unit of an egg cell (EC), synergids (SY), and central cell(CC). Bars: A 50 m, B 100 mA transgenic line with bright fluorescence in allsomatic tissues was analyzed for expression of GFP ingametes and zygotes. In female gametophytes the35S:gfp construct was expressed. Egg cells, synergids,and central cells showed GFP fluorescence (Fig. 1B),whereas no fluorescence was detected in transgenic malegametes. This differential expression pattern of the35S:gfp construct offers the opportunity to determine thetiming of male genome activation with respect to thetransgene. Following in vitro fusion of non transgenicegg cells with transgenic sperm cells, transcription of thetransgene was induced early after fertilization and GFPcould be detected in zygotes (S. Scholten, unpublishedresults). Analysis is in progress in our laboratory todetermine the onset of RNA and protein synthesis in eggcells fertilized with transgenic sperm cells.These results show the activity of the 35S promoterconstruct in egg cells, central cells, and zygotes soonafter fertilization. This opens the possibility of designingnew experiments and expressing other transgenes undercontrol of this promoter construct during fertilization andearly zygotic embryogenesis.Chemical inducible expression of transgenesGenes with a critical role in development are of specialinterest for functional analysis in sexual plant reproduction.A major limitation of constitutive promoters is that theycannot be used to investigate genes whose alteredexpression (by a sense or antisense transgene) has delete-rious effects on the plant cells, thereby blocking plantregeneration. These deleterious effects are very likely forgenes essential to early development. Chemically inducibleexpression systems offer a solution to this problem. Themain advantages of chemically inducible expression arethe potential to express gene products that interfere withregeneration, growth or reproduction; to express geneproducts at different times during development and; toobserve the clear correlation between the induction of atransgene and occurrence of an altered phenotype (Gatzand Lenk 1998).The general strategy to establish chemically inducibleexpression systems for plants is the transfer of regulatoryelements from non-plant sources. The first chemicallyregulated system, developed by Gatz and Quail (1988),is based on the bacterial tetracyclin repressor. Since then,integration of regulatory domains of animal steroidhormone receptors has led to development of systemsbased on transcriptional activation (e.g. Aoyama andChua 1997; Bhner et al. 1999; Bruce et al. 2000; forreview see Zuo and Chua 2000).These systems consist of a constitutively expressed,activating transcription factor whose activity can becontrolled by a ligand. Transcription of the transgene iscontrolled by an inducible promoter with cis-elementsfor the transcription factor. We used the estrogen receptor-based system described by Bruce et al. (2000) to generatestable transformed maize plants by microprojectile bom-bardment. The activator construct, the promoter construct,and the selection marker where co-transformed on differentplasmids. The transformation frequency of these experi-ments is shown in Table 1. Approximately every fifthtransgenic regenerant showed inducible expression.Northern blot analysis revealed the induction level of thesystem to be dependent on ligand concentration (Fig. 2B).The induction level of a transgenic plant reaches about50% of the transcript level of a plant with a high expres-sion level of the same GFP construct under control of the35S promoter (Fig. 2A). Basic activity of the system wasnot be detected even when the exposure time, as shownin Fig. 2A, was increased 48-fold. With these characteris-tics, the inducible system is well suited for overexpres-sion of genes whose products interfere with regenerationas for expression of antisense RNA. Analyses are in pro-gress in our laboratory to test the function of this systemin gametes and zygotes.ProspectsTransient expression studies after microinjection offoreign genes into egg cells and zygotes, as well as38Table 1 Transformation frequencies with an inducible expressionsystem. Transgenic plants were generated by particle bombardmentof immature embryos. Analysis was performed by nothern-blot-hybridization. See text for further explanationsPrimary Transgenic Inducibleexplants plants plantsTotal number 1881 71 14Frequency (%) 100 3.7 0.7Fig. 2A, B Ligand-regulated expression in transgenic maize plants.Leaf material was excised and cultured 48 h in water alone () orin water with ligand (+). Afterwards total RNA was isolated,separated, immobilized and hybridized with a gfp-specific probe.A 1/2 control plant with strong, constitutive gfp expression. xH99Non-transgenic control plant. 4/1, 4/3 transformants with gfpunder control of the inducible expression system. B Ligandconcentration-dependent expression level in plant 4/3. Ligandconcentrations are indicated. Below 28S rRNA is shown as loadingcontrolanalyses of the expression of stable integrates, showedthat these cells are able to express transgenes. Thisprovides the opportunity for functional analysis usingtransgenic approaches during fertilization and very earlyzygotic development. Transcription factors expressed inegg cells and cell cycle regulators might both have acritical role during fertilization, further development ormorphogenesis. They are thus interesting candidates forantisense and overexpression studies. If the inducibleexpression systems are functional in gametes and zygotes,it could greatly enhance experimental output or evenmake studies possible when altered expression of a givengene is lethal.In vitro fertilization and culture systems enable directobservation and monitoring of the development of indi-vidual cells. Combining this option with the expressionof GFP-based marker genes for subcellular structuresenables new stategies to analyze cytological characteristicsduring fertilization, and during early zygotic and endo-sperm development. Using introduced GFP-based markersfor cytoskeletal components is of high interest, since theplant cytoskeleton has crucial functions in cellularprocesses essential for cell morphogenesis and develop-ment (Kost et al. 1999). Double-labeling experimentswith spectral GFP variants (Yang et al. 1998) or therecently isolated red fluorescent protein (Matz et al. 1999)might enable analysis of dynamic changes and the inter-actions of actin filaments and microtubules. Once estab-lished through transgenic maize plants, GFP-based cyto-logical markers might be valuable for correlating expres-sion data with specific developmental stages, e.g. cellcycle phases, through visualisation of microtubularstructures. 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