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Isolation of Fucus serratus Gametes and Cultivation of the Zygotes
齿缘墨角藻配子的分离和合子的培养   

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Abstract

Zygotes of the Fucale species are a powerful model system to study cell polarization and asymmetrical cell division (Bisgrove and Kropf, 2008). The Fucale species of brown algae grow in the intertidal zone where they reproduce by releasing large female eggs and mobile sperm in the surrounding seawater. The gamete release can be induced from sexually mature fronds in the laboratory and thousands of synchronously developing zygotes are easily obtained. In contrast to other eukaryotic models, such as land plants (Brownlee and Berger, 1995), the embryo is free of maternal tissues and therefore readily amenable to pharmacological approaches. The zygotes are relatively large (up to 100 µm in diameter), facilitating manipulations and imaging studies. During the first hours of zygote development, the alignment of the axis to external cues such as light is labile and can be reversed by light gradients from different directions. A few hours before rhizoid emergence, the alignment of the axis and the polarity are fixed and the cells germinate accordingly. At this stage the zygotes are naturally attached to the substratum through the secretion of cell wall adhesive materials (Kropf et al., 1988; Hervé et al., 2016). The first cell division occurs about 24 h after fertilisation and the early embryo is composed of only two cell types that differ in size, shape and developmental fates (i.e., thallus cells and rhizoid cells) (Bouget et al., 1998). The embryo can be successfully cultivated in the laboratory for a few more days (4 weeks maximum) and has an invariant division pattern during the early stages, which allows cell lineages to be traced histologically.

Keywords: Asymmetric cell division(不对称细胞分裂), Developmental biology(发育生物学), Brown algae(棕色藻类), Fucus serratus(齿缘墨角藻), Zygotes(合子), Embryogenesis(胚胎发生)

Background

This protocol provides instructions for the isolation of male and female gametes of Fucus spp. used for in vitro fertilisation and discusses how to monitor the development of the resulting zygotes and early embryos. These instructions are for the use of the dioecious species Fucus serratus, but they can be readily adapted to a monoecious Fucale species.

Materials and Reagents

  1. Razor blade (Gilette)
  2. Black flat base
    Note: This can be a black paper base.
  3. Paper towel (Lucart Professional, catalog number: 864043 )
  4. 100 μm nylon filter (Sigma-Aldrich, catalog number: NY1H00010 )
  5. Slides
  6. Petri dishes (SARSTEDT, catalog number: 82.1472.001 )
  7. Sexually mature plants of Fucus serratus, males and females
    Note: Marine Research Institutes are sometimes equipped with facilities that allow the ordering and sending of such material (see for instance the EMBRC-France website, http://www.embrc-france.fr/en) The algae can be transported in moist tissues for a time preferentially no longer than 2 days.
  8. Approximately 1 L of natural seawater
    Note: Alternatively artificial seawater can be prepared or purchased (Sigma-Aldrich, catalog number: S9883 ).

Equipment

  1. Beaker of approximately 250 ml (Fisher scientific)
  2. Desk lamp
  3. Fridge or cold room
  4. Optical microscope equipped with 20x and/or 40x objectives (Olympus)
  5. Thermostatic chamber at 13 °C (Pol-Eko Aparatura)
  6. White light, ideally 90 µmol m-2 sec-1 (Mazda)
  7. Hermetic black chamber (optional)

Procedure

  1. Isolation of Fucus serratus gametes and fertilisation
    1. Collect female and male plants of Fucus serratus on the shore (Figure 1, step A1).
      During a certain period of the year (winter time for European coasts), the thallus tips of the Fucale species are swollen into receptacles that contain the fertile conceptacles. On the shore, male plants can be recognized by the orange colour of their conceptacles, due to a carotenoid pigment present in the sperm chromoplasts. The female plants are brown-green in colour (Figure 1B1 and 1B2). Sort female and male plants. To do this, section by hand a conceptacle, mount sections on a microscope slide with a drop of seawater and examine by light microscopy. Typical pictures of female and male conceptacles are shown (Figure 1B3 and 1B4). Isolate all sexually mature fronds in two distinct batches (i.e., male/female).
    2. Wash in seawater to remove epiphytes and remaining sand and wash briefly with tap water (Figure 1, step A2).
    3. Spread all pieces on paper towel (Figure 1, step A3) making layers, and roll the whole such as for a sushi maki (Figure 1B5 and 1B6). Store at 4 °C in darkness for at least one week.
    4. To release the female gametes, unroll the paper towels and transfer the mature fronds into a beaker containing seawater (Figure 1, step A4). Incubate one hour in daylight or under artificial light at room temperature. The oogonium, which contains eight individual eggs in F. serratus, will be released first, rapidly rupturing to discharge the eggs. Examine by light microscopy.
      Note: The stress induced by the abrupt light and osmotic changes (i.e., switching samples from darkness to daylight and back in seawater) favour the gamete release.


      Figure 1. Overview of the key stages in the isolation of Fucus gametes and cultivation of the zygotes. A. The key stages are listed on the left side. B. Representative pictures are shown on the right side for: B1 and B2. Female and male Fucus serratus plants; B3 and B4. Cross sections of their respective gamete-containing conceptacles; B5 and B6. The conditioning of the mature fronds for storage; B7. Typical 4-celled zygotes obtained after 48 h following fertilisation.

    5. At this stage the eggs will rapidly settle at the bottom of the beaker. After 2 h incubation gently remove some seawater with a pipette to concentrate the eggs. Filter the remaining solution containing the eggs using a 100 µm nylon mesh filter to remove aggregates. Place one or two small pieces of male fronds in contact with the newly released eggs (Figure 1, step A5). The male gametes exhibit negative phototropism, therefore placing the beaker in full daylight or under artificial light on a black flat base will favour the sperm movement toward the eggs. The female cells release a sexual pheromone to attract the sperm. Fertilisation will occur within 15 min of mixing sperm with oospheres. After 30 min, examine the release and vitality of the male gametes by light microscopy (Video 1).

      Video 1. Fertilisation of a Fucus serratus egg by sperm of the same species. The male gametes are motile and clearly visible as small cells actively swimming in the vicinity of the immotile female egg. Flagella on the male gametes can be detected. The egg is approximately 80 µm large.

    6. After one hour, wash twice by gently removing some of the seawater with a pipette and adding fresh seawater. Filter the remaining solution using a 100 µm nylon mesh filter. Allow the zygotes to grow by placing the beaker at 13 °C with a photoperiod of 12 h of light (90 µmol m2 sec-1) and 12 h of darkness (Figure 1, step A6). The zygotes will naturally adhere to their substratum through the secretion of cell wall adhesive materials.
      Note: To ease future examination, the zygotes can be directly cultivated on microscope slides. To do this, concentrate the zygotes to an appropriate density and drop 250 µl of the solution on the slides. The use of polylysine-coated microscope slides will allow the zygotes and early embryos to stay firmly attached.

  2. Monitoring development of Fucus serratus zygotes and early embryos
    1. Typical growth conditions are 13 °C with a photoperiod of 12 h of light (90 µmol m2 sec-1) and 12 h of darkness. Different stages of early development can be easily observed by light microscopy. The first cell division occurs around 24 h after fertilisation and divides the embryo in two cell types having distinct shapes and fates (Figure 2).


      Figure 2. Early development of Fucus serratus zygote and embryo. A. Fucus eggs are spherical in shape with no obvious asymmetries. B. Polarization is first manifested morphologically, around 20 h after fertilisation, when zygotes elongate and produces a bulge on one hemisphere, the rhizoid. C. When the first cell division occurs, about 24 h after fertilisation, it bisects the zygote transversally into two asymmetrical cells with distinct developmental fates: the apical thallus cell and the rhizoid basal cell. D. During the following cell divisions, the rhizoid cell elongates by apical growth whereas the thallus cell expands by diffuse growth.

    2. Regularly change the surrounding seawater to renew nutrients and prevent desiccation. Cultivation in Petri dishes will require the seawater (15-50 ml) to be changed once a week.
    3. It is possible to control the orientation of the polar axis formation and fixation by applying a light vector. To do this a unilateral light (L1) should be used (a classical light bulb can be used and should not be positioned above the Petri dish but parallel to the bench base). The rhizoid cell will develop in the opposite direction of the light vector L1 (Figure 3A). In normal conditions, the polarization axis is labile during the first 10 h of development and can therefore be redirected with the use of a second unidirectional light gradient (L2), orientated 180° to the first (Figures 3B and 3C). This property allows ready study of the mechanisms controlling polarization by treatment with pharmacological agents. The use of a hermetic black chamber, equipped with a unilateral bulb light, reduces the impact of possible external and uniform light.
    4. The embryos can be easily grown for one week in the laboratory. Beyond this stage the mature embryos are difficult to maintain in lab culture conditions (4 weeks maximum). Unlike other models of brown algae, such as the Ectocarpus filamentous alga, the life cycle of F. serratus cannot be reproduced in the laboratory.


      Figure 3. Control of the orientation of the polar axis formation and fixation. A. Starting just after fertilization (AF), the zygotes are cultivated in a unilateral light direction (L1). The rhizoid cell will develop in the opposite direction of L1. B. Zygotes are cultivated in the unilateral light direction (L1) during the first hours AF. Before axis fixation (approximately 8 h AF), a second unilateral light vector (L2) is applied, oriented 180° from L1. The rhizoid cell develop in the opposite direction of L2. C. If the switch of light directions is applied after axis fixation, the rhizoid cell will develop in the opposite direction of the first light source.

Data analysis

Fucus zygotes develop synchronously and the protocol listed above allows the observation of hundreds of individuals at the same time. For data analysis (i.e., morphological results from pharmacological studies) we recommend performing three individual biological replicates. For each replicate a minimum of 30-100 zygotes should be observed. Statistical tests should be applied such as Student’s t-tests (Hervé et al., 2016).

Acknowledgments

A brief description of this protocol has been recently reported in Hervé et al., 2016 and was mainly based on previously described methods (Kropf et al., 1988; Bouget et al., 1998). We acknowledge funding from the Brittany Region (grant ARED_8979 ECTOPAR).

References

  1. Bisgrove, S. and Kropf,  D. L. (2008). Asymmetric cell divisions: zygotes of fucoid algae as a model system. In: Verma, D. and Hong, Z. (Eds.). Cell Division Control in Plants. Springer pp: 323-341.
  2. Bouget, F. Y., Berger, F. and Brownlee, C. (1998). Position dependent control of cell fate in the Fucus embryo: role of intercellular communication. Development 125(11): 1999-2008.
  3. Brownlee, C. and Berger, F. (1995). Extracellular matrix and pattern in plant embryos: on the lookout for developmental information. Trends Genet 11(9): 344-348.
  4. Hervé, C., Siméon, A., Jam, M., Cassin, A., Johnson, K. L., Salmeán, A. A., Willats, W. G., Doblin, M. S., Bacic, A. and Kloareg, B. (2016). Arabinogalactan proteins have deep roots in eukaryotes: identification of genes and epitopes in brown algae and their role in Fucus serratus embryo development. New Phytol 209(4): 1428-1441.
  5. Kropf, D. L., Kloareg, B. and Quatrano, R. S. (1988). Cell wall is required for fixation of the embryonic axis in Fucus zygotes. Science 239(4836): 187-190.

简介

Fucale物种的合作伙伴是研究细胞极化和不对称细胞分裂的强大模型系统(Bisgrove和Kropf,2008)。棕色藻类的Fucale种类在潮间带中生长,通过在周围海水中释放大的雌性卵和移动精子繁殖。配子释放可以从实验室的性成熟叶子诱导,并且容易获得成千上万的同步发育的合子。与其他真核生物模型(如陆地植物(Brownlee和Berger,1995))相反,胚胎没有母体组织,因此很容易接受药理学方法。受精卵相对较大(直径可达100μm),便于操作和成像研究。在合子开发的第一个小时,轴与光线等外部线索的对准不稳定,可以通过不同方向的光梯度反转。在芽根出现前几小时,轴和极性的对齐是固定的,并且细胞相应地发芽。在这个阶段,合子通过细胞壁粘合剂材料的分泌(Kropf等人,1988;Hervé等人,2016)自然地附着在基质上。第一个细胞分裂发生在受精后约24小时,早期胚胎仅由两种细胞类型组成,其尺寸,形状和发育命运(,thallus细胞和根际细胞)不同(Bouget 等人,1998)。胚胎可以在实验室中成功培养数日(最多4周),并且在早期阶段具有不变的分裂模式,可以使组织学上追踪细胞谱系。
【背景】该协议提供了隔离Fucus spp的雄性和雌性配子的说明。 用于体外受精,并讨论如何监测所产生的受精卵和早期胚胎的发育。 这些说明是为了使用雌雄异株(Fucus serratus),但是它们可以很容易地适应于一种独特的Fucale物种。

关键字:不对称细胞分裂, 发育生物学, 棕色藻类, 齿缘墨角藻, 合子, 胚胎发生

材料和试剂

  1. 剃刀刀片(Gilette)
  2. 黑色平底座
    注意:这可以是一个黑色纸张基地。
  3. 纸巾(Lucart Professional,目录号:864043)
  4. 100微米尼龙过滤器(Sigma-Aldrich,目录号:NY1H00010)
  5. 幻灯片
  6. 培养皿(SARSTEDT,目录号:82.1472.001)
  7. 黑颈草属性成熟植物,男性和女性
    注意:海洋研究所有时可以配备允许订购和发送这种材料的设施(例如参见EMBRC-France网站, http://www.embrc-france.fr/en )藻类可以在潮湿的组织中运输优先不超过2天的时间。
  8. 约1升天然海水
    注意:或者可以制备或购买人造海水(Sigma-Aldrich,目录号:S9883)。

设备

  1. 烧杯约250毫升(费雪科学)
  2. 台灯
  3. 冰箱或冷藏室
  4. 光学显微镜配备20x和/或40x目标(Olympus)
  5. 13°C的恒温室(Pol-Eko Aparatura)
  6. 白光,理想的是90μmol/ m 2,上一次(马自达)
  7. 气密黑室(可选)

程序

  1. 配子和受精的分离
    1. 在岸上收集 fucus serratus 的雌性和雄性植物(图1,步骤A1)。
      在一年的某一时期(欧洲海岸的冬季),Fucale物种的thallus尖端肿胀成容纳有丰富概念的容器。在岸上,由于精子色素中存在类胡萝卜素,所以雄性植物可以被它们的概念的橙色所识别。雌性植物是棕绿色的(图1B1和1B2)。排列女性和男性植物。为了做到这一点,手工绘制一个概念,用一滴海水将显微镜载玻片上的部分放置,并通过光学显微镜检查。显示了女性和男性概念的典型照片(图1B3和1B4)。在两个不同的批次中分离所有性成熟的叶状体(,即男性/女性)。
    2. 在海水中洗涤以除去附生植物和剩余的沙子,并用自来水短暂洗涤(图1,步骤A2)。
    3. 将所有片材放在纸巾上(图1,步骤A3)制作层,并将其全部卷成寿司(图1B5和1B6)。在黑暗中在4°C储存至少一周。
    4. 要释放雌配子,展开纸巾,并将成熟叶子转移到含海水的烧杯中(图1,步骤A4)。在室温下在白昼或人造光照下孵育1小时。 oogonium,其中包含在F中的八个单独的卵。 serratus 将首先释放,迅速破裂以排卵。通过光学显微镜检查。
      注意:由突然的光和渗透变化引起的应力(即将样品从黑暗切换到日光并在海水中回流)有利于配子释放。


      图1.分离“fucus”配子和合作伙伴培育的关键阶段概述。 A.关键阶段列在左侧。 B.代表性图片显示在右侧:B1和B2。女性和男性镰刀菌属植物; B3和B4。它们各自配子的概念的横截面; B5和B6。成熟叶片的调理保存; B7。在受精48小时后获得典型的4细胞受精卵。

    5. 在这个阶段,鸡蛋将在烧杯的底部快速沉降。孵育2小时后,用移液管轻轻取出一些海水,以浓缩蛋。使用100μm尼龙网过滤器过滤剩余的含有鸡蛋的溶液,以去除聚集体。将一个或两个小块的雄性叶子与新释放的鸡蛋接触(图1,步骤A5)。男性配子表现出负向光性,因此将烧杯置于充分的日光下或在人造光下放置在黑色的平底上,将有利于精子向卵移动。女性细胞释放一种性信息素来吸引精子。施肥将在精子混合15分钟内发生。 30分钟后,检查雄配子的释放和活力
      Video 1. Fertilisation of a Fucus serratus egg by sperm of the same species. The male gametes are motile and clearly visible as small cells actively swimming in the vicinity of the immotile female egg. Flagella on the male gametes can be detected. The egg is approximately 80 µm large.

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    6. 一小时后,用移液管轻轻取出一些海水并加入新鲜的海水,洗两次。使用100μm尼龙网过滤器过滤剩余的溶液。通过将烧杯放置在13℃,光照时间为12小时光照(90μmol/ m 2)以上12小时黑暗(允许)图1,步骤A6)。受精卵通过分泌细胞壁粘合剂材料自然会粘附到底层。
      注意:为了方便将来的检查,合子可以直接在显微镜载玻片上进行培养。为了做到这一点,将合子集中到适当的密度,并将250μl的溶液放在载玻片上。使用聚赖氨酸涂层的显微镜载玻片将允许合子和早期胚胎保持牢固连接。

  2. 监测 fucus serratus 合约和早期胚胎的开发
    1. 典型的生长条件为13℃,光照时间为12小时光(90μmol/平方米),12小时黑暗。早期发育的不同阶段可以通过光学显微镜容易地观察到。第一个细胞分裂发生在受精后约24小时,并将胚胎分成具有不同形状和命运的两种细胞类型(图2)。


      图2. fucus serratus 合子和胚胎的早期发展。 A. Fucus 鸡蛋是球形的,没有明显的不对称性。 B.极化首先在形态上表现出来,受精后约20小时,当合子伸长并在一个半球上产生隆起时,即根茎。当第一次细胞分裂发生时,受精后约24小时,将受精卵横向分成两个具有不同发育命运的不对称细胞:顶端thallus细胞和根际细胞。 D.在以下细胞分裂期间,根尖细胞由顶端生长延伸,而Thallus细胞通过扩散生长扩张。

    2. 定期更换周围海水,更新营养,防止干燥。培养皿培养将需要每周更换一次海水(15-50毫升)。
    3. 可以通过施加光矢量来控制极轴形成和固定的取向。要做到这一点,应该使用单边光(L1)(可以使用经典的灯泡,不应位于培养皿上方,而应平行于台架)。根状细胞将沿着光矢量L1的相反方向发展(图3A)。在正常条件下,偏振轴在显影的第一个10小时期间是不稳定的,因此可以使用第二单向光梯度(L2)重定向,朝向第一个(图3B和3C)180°。该属性允许通过用药理学剂处理来控制极化的机理。使用装有单面灯泡的密封黑色室,减少了可能的外部和均匀光线的影响。
    4. 胚胎可以在实验室中容易地生长一周。在这个阶段之后,成熟的胚胎在实验室培养条件下难以维持(最多4周)。与其他型号的棕色藻类不同,如Ectocarpus 丝状藻类,F的生命周期。 serratus 不能在实验室中复制。


      图3.极轴形成和固定方向的控制。 A.刚刚施肥(AF)后,合子在单向光照(L1)下栽培。 Rhizoid细胞将沿L1的相反方向发育。在第一个小时AF期间,接受合作的单方向(L1)种植。在轴固定(约8小时AF)之前,施加第二单向光矢量(L2),从L1定向180°。 Rhizoid细胞沿L2的相反方向发育。 C.如果在轴固定之后施加光线方向的切换,则根尖细胞将沿与第一光源相反的方向发展。

数据分析

合肥同步发展,上述协议允许同时观察数百人。对于数据分析(即,,药理学研究的形态学结果),我们建议执行三个单独的生物重复。对于每个重复,应至少观察30-100个合子。应该采用统计学测试,例如,Student's tests(Hervé等人,2016)。

致谢

最近在Hervé等人,2016中简要介绍了该协议,主要是基于以前描述的方法(Kropf等人,1988; Bouget et al。,1998)。我们承认布列塔尼地区的资助(拨款ARED_8979 ECTOPAR)。

参考

  1. Bisgrove,S。和Kropf,DL(2008)。不对称细胞分割:岩藻类藻类的合作伙伴作为模型系统。在:Verma,D.and Hong,Z.(Eds。)。植物细胞分裂控制。 Springer pp:323-341。
  2. Bouget,FY,Berger,F.and Brownlee,C。(1998)。  位置依赖的控制细胞命运在 Fucus 胚胎中:细胞间通讯的作用 发展 125(11):1999-2008。 br />
  3. Brownlee,C.和Berger,F.(1995)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/7482785”target =“_ blank” >植物胚胎中的细胞外基质和模式:寻找发育信息。趋势Genet 11(9):344-348。
  4. Hervé,C.,Siméon,A.,Jam,M.,Cassin,A.,Johnson,KL,Salmeán,AA,Willats,WG,Doblin,MS,Bacic,A.and Kloareg,B.(2016) ; 阿拉伯半乳聚糖蛋白质在真核生物中有深刻的基础:鉴定基因和表位在棕色藻类中,它们在胚胎发育中的作用。新的Phytol 209(4):1428-1441。
  5. Kropf,DL,Kloareg,B.and Quatrano,RS(1988)。< a class =“ke-insertfile”href =“https://www.ncbi.nlm.nih.gov/pubmed/3336780”target = “_blank”>需要细胞壁来固定胚胎轴在 合子中。 科学 239(4836):187-190。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Siméon, A. and Hervé, C. (2017). Isolation of Fucus serratus Gametes and Cultivation of the Zygotes. Bio-protocol 7(14): e2408. DOI: 10.21769/BioProtoc.2408.
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