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Shoot Apical Meristem Size Measurement
顶端分生组织大小的测量   

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The Plant Journal
Feb 2016

Abstract

The shoot apical meristem (SAM) is a collection of cells that continuously renew themselves by cell division and also provide cells to newly developing organs. It has been known that CLAVATA (CLV) 3 peptide regulates a transcription factor WUSCHEL (WUS) to keep numbers of undifferentiated cells constant and maintain the size of the SAM. The interactive feedback control of CLV3 and WUS in a non-cell autonomous signaling cascade determines stem cell fate (maintenance of pluripotency or, alternatively, differentiation into daughter cells) in the SAM. Ca2+ is a secondary messenger that plays a significant role in numerous signaling pathways. The signaling system connecting CLV3 binding to its receptor and WUS expression is not well delineated. We showed that Ca2+ is involved in CLV3 regulation of the SAM size. One of the approaches we used was measuring the size of the SAM. Here we provide a detailed protocol on how to measure Arabidopsis SAM size with Nomarski microscopy. The area of the two-dimensional dome representing the maximal ‘face’ of the SAM was used as a proxy for SAM size. Studies were done on wild type (WT) Arabidopsis in the presence and absence of a Ca2+ channel blocker Gd3+ and the CLV3 peptide, as well on genotypes that lack functional CLV3 (clv3) or a gene encoding a Ca2+-conducting ion channel (‘dnd1’).

Keywords: Arabidopsis (拟南芥), Shoot apical meristem (顶端分生组织), Shoot development (枝稍发育), Cell signaling (细胞信号), Seedlings (幼苗)

Background

Nomarski microscopy is widely used to study Arabidopsis SAM size. Other microscopy techniques for SAM observation are time consuming and require embedding tissue in resin and then sectioning or even more sophisticated microscopy. Nomarski microscopy, along with tissue clearing techniques is fast and convenient for whole tissue imaging. Published methods on SAM size measurement with Nomarski microscopy are often briefly described. Here, we provide a modified protocol with a detailed step by step guide including steps from dissecting Arabidopsis SAM tissues, through sample preparation for Nomarski microscopy, and SAM size measurement.

Materials and Reagents

  1. 3 x 4 cell culture multi-well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150628 )
  2. Parafilm
  3. Razor blade
  4. Glass slide (75 x 38 mm) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-550B )
  5. Coverslips (18 x 18 mm) (thickness: 0.17 mm) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S17521 )
  6. Arabidopsis thaliana wild type (ecotype Columbia), dnd1 mutant (At5g15410), clv3 mutant (At2g27250)
  7. Synthetic CLV3 peptide RTVPhSGPhDPLHH3 (GenScript, Piscataway, NJ)
  8. Murashige and Skoog salts (MS) (Caisson Laboratories, catalog number: MSP01-10LT )
  9. Gadolinium(III) chloride (GdCl3) (Sigma-Aldrich, catalog number: 439770 )
  10. Ethanol (Sigma-Aldrich, catalog number: 459836 )
  11. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  12. MES buffer (pH 5.7) (Caisson Laboratories, catalog number: M009-100GM )
  13. Sterilized water
  14. Tris (Sigma-Aldrich, catalog number: 252859 )
  15. Acetic acid (Thermo Fisher Scientific, Fisher Scientific, catalog number: A38S-500 )
  16. Chloral hydrate (Sigma-Aldrich, catalog number: C8383 )
  17. Glycerol (Thermo Fisher Scientific, catalog number: 17904 )
  18. Plant culture medium (see Recipes)
  19. Fixing solution (see Recipes)
  20. Clearing solution (see Recipes)

Equipment

  1. Fridge
  2. Shaker
  3. Growth chamber for growing plants (100 µmol m-2 sec-1 white light for 16 h and dark for 8 h, 23 °C)
  4. Tweezers
  5. Dissecting microscope
  6. Microscope equipped with Nomarski optics (Nikon Instrument, model: MICROPHOT-FX )

Software

  1. Infinity analyze program (Lumenera, Ottawa, Canada)

Procedure

  1. Arabidopsis seedling culture and treatments
    1. Add 2 ml ½ MS liquid media to each well of a multi-well plate.
    2. Surface sterilize all seeds and place 1 seed into each well of the multi-well plate.
    3. Stratify the seeds by placing the multi-well plate at 4 °C for 3 days to break dormancy.
    4. After stratification, take the multi-well plate out from the 4 °C fridge and add different treatments to each well. In our experiment, we added CLV3 ligand alone (1 μM final concentration), Ca2+ channel blocker GdCl3 alone (150 μM final concentration) and CLV3 with GdCl3 together (at final concentrations of 1 and 150 μM, respectively) to ½ MS liquid media.
      Note: Because CLV3 peptide was dissolved in water, the control group should be water and should be the same amount as the CLV3 treatment group. The small amount of water added (in our experiment was 2 μl) should not have any osmotic stress on seedlings. 
    5. Put the multi-well plate containing seedlings on a rotatory shaker (180 rpm) in a growth chamber (100 µmol m-2 sec-1 white light for 16 h and dark for 8 h, 23 °C). We define this day as our beginning day of experiment (day 0).

  2. Seedling preparation for dissection
    1. After 7 days of growth in the growth chamber, gently take out individual seedlings one at a time from the multi-well plate with a tweezer and put it on a piece of paper towel for 3 sec to absorb excessive media.
    2. Remove the individual seedling from the paper towel with a tweezer and lay it sideways on a piece of Parafilm under a dissecting microscope.
    3. Remove the roots, cotyledons and the oldest leaf primordia with a sharp razor blade.
    4. To prevent morphology change, quickly (but gently) move dissected tissue with a tweezer into a well of the same plate containing only freshly made fixing solution and incubate overnight at room temperature.
    5. Remove fixing solution the next day and replace with 90% ethanol. Put the plate on a shaker at room temperature for up to 1 h (40 rpm).
    6. Remove 90% ethanol and replace with 70% ethanol. Put the plate on a shaker at room temperature for up to 1 h (40 rpm).
    7. Remove dissected tissue one at a time from the 70% ethanol and place on a glass slide with a drop of freshly made clearing solution.
    8. Observe under a microscope equipped with Nomarski optics (MICROPHOT-FXA; Nikon, Tokyo, Japan).
      Note: The reaction of clearing solution is fast (~5 min). It is best to look at 1 to 2 samples at a time and take pictures as soon as the SAM is observed under the microscope. After a while the dome of the SAM will be hard to distinguish from surrounding tissues. The rest of the seedlings can be kept in 70% ethanol before observing under microscope.

  3. SAM area measurement.
    1. Open Infinity analyze software program.
    2. Select ‘capture’ to take photographs. Record individual seedling images at the focal plane corresponding to the median optical section of the SAM.
    3. Draw a straight line between the basal edges of two opposing leaf primordia on each side of the SAM dome. This line represents the base of the SAM dome (Figure 1). A perpendicular line was generated connecting the midpoint of the SAM base to the top of the SAM dome (Figure 1C).
    4. Select ‘Area perimeter’ for SAM size measurement. Circle a closed SAM shape area based on the two lines described in step C3 by freehand drawing. The area of the SAM is determined by calculating the area above the straight line that represented the width of the base of the SAM. The program will automatically calculate the area and the perimeter.
       

      Figure 1. 7-d-old Arabidopsis SAM area under a microscope equipped with Nomarski optics. A. Wild type. B. DEFENSE NO DEATH 1 (dnd1) mutant, which lacks a functional cyclic nucleotide gated cation channel isoform 2 (CNGC2) polypeptide and (C) clv3 mutant. Yellow triangles indicate the basal edges of the SAM dome. The red line that connects the 2 yellow triangles represents the base of the SAM dome. The perpendicular red line connects the midpoint of the SAM base to the top of the SAM dome.

Data analysis

For comparison between SAM size of wild type, dnd1 and clv3 mutant seedlings (see Figure 1 and Figure 2A), at least 15 seedlings of each genotype were measured. For calcium channel blocker effects on endogenous CLV3 in wild type (Figure 2B) and exogenous CLV3 effects on clv3 mutant, at least 10 seedlings were measured for each treatment (Figure 2C). ANOVA analysis was used to evaluate means separation. For Figures 2A and 2B, an asterisk or two asterisks above the bar representing a genotype or treatment indicate SAM size was significantly different (at P < 0.05 or P < 0.01, respectively) than control (WT). For Figure 2C, ANOVA comparisons are indicated by brackets.


Figure 2. Ca2+ signaling interacts with CLV3 control of SAM area. (a). SAM area of 7-d-old WT, dnd1 and clv3 seedlings. (b). WT seedlings grown on standard ½ MS liquid medium (Control), or medium supplemented with Gd3+ µM on day 3 or on day 0 (the day the experiment starts). (c). clv3 seedlings grown on standard medium were treated with water (control), CLV3, or CLV3 and Gd3+. CLV3 and Gd3+ were applied on day 0. ANOVA analysis was used to evaluate means separation. All seedlings shown in Figure 2 are 7-d-old seedlings. For (a) and (b), an asterisk or two asterisks above the bar representing a genotype or treatment indicate SAM size was significantly different (at P < 0.05 or P < 0.01, respectively) than control (WT). For (c), ANOVA comparisons are indicated by brackets.

Recipes

  1. Plant culture medium
    Dissolve 2 g sucrose, 0.433 g ½ MS salt, and 2 ml MES in 200 ml MiliQ water, and then adjust the pH to 5.7 with Tris. Autoclave medium
  2. Fixing solution
    Acetic acid:absolute ethanol (1:9, v/v)
  3. Clearing solution
    Chloral hydrate, glycerol and water (8:1:2, w/v/v)

Acknowledgments

This work was supported by National Science Foundation award 1146827 (to G.A.B). This protocol was adapted and modified from Fiers et al., (2006), Ohyama et al., (2009), Carles et al., (2010) and Dr. Miguel Aguilar.

References

  1. Aguilar M. Identifying features of mutant seeds using nomarski microscopy (Gene one).
  2. Carles, C. C., Ha, C. M., Jun, J. H., Fiume, E. and Fletcher. J. C. (2010). Analyzing shoot apical meristem development. Plant Development Biol 655:105-29.
  3. Fiers, M., Golemiec, E., Schors, R., Van, Der., Geest, L., Van, D., Li, K. W., Stiekema, W. J. and Liu, C. M. (2006). The CLAVATA3 / ESR motif of CLAVATA3 is functionally independent from the nonconserved flanking sequences. Plant Physiol 141(4): 1284-1292.
  4. Ohyama, K., Shinohara, H., Ogawa-Ohnishi, M. and Matsubayashi, Y. (2009). A glycopeptide regulating stem cell fate in Arabidopsis thaliana. Nat Chem Biol 5(8): 578-580.

简介

芽顶端分生组织(SAM)是通过细胞分裂不断更新自身的细胞的集合,并且还向新发育的器官提供细胞。已知CLAVATA(CLV)3肽调节转录因子WUSCHEL(WUS)以保持未分化细胞的数量恒定并维持SAM的大小。在非细胞自主信号级联中的CLV3和WUS的交互反馈控制确定SAM中的干细胞命运(多能性的维持,或者,分化成子细胞)。 Ca 2 + 是在许多信号传导途径中起重要作用的第二信使。连接CLV3结合其受体和WUS表达的信号系统没有很好地描绘。我们显示Ca 2 + 参与SAM大小的CLV3调节。我们使用的方法之一是测量SAM的大小。在这里,我们提供了一个详细的协议,如何用Nomarski显微镜测量拟南芥SAM大小。将代表SAM的最大"面"的二维圆顶的面积用作SAM大小的代表。在存在和不存在Ca 2+通道阻断剂Gd 3+和sLV 3肽的情况下对野生型(WT)拟南芥进行研究。 ,以及缺乏功能性CLV3( clv3 )或编码Ca 2+ 2+导电离子通道的基因('dnd1 ')的基因型。
关键字: 拟南芥,射击顶端分生组织,苗发育,细胞信号,幼苗

> Nomarski显微镜广泛用于研究拟南芥SAM大小。用于SAM观察的其他显微技术是耗时的,并且需要将树脂嵌入树脂中,然后切片或甚至更复杂的显微镜。 Nomarski显微镜,连同组织清除技术是快速和方便的整个组织成像。通常使用Nomarski显微镜对SAM尺寸测量的发布方法进行简要描述。在这里,我们提供了一个修改的协议,详细的一步一步指南,包括解剖拟南芥SAM组织,通过样品制备的Nomarski显微镜和SAM大小测量。

关键字:拟南芥, 顶端分生组织, 枝稍发育, 细胞信号, 幼苗

材料和试剂

  1. 3×4细胞培养多孔板(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:150628)
  2. parafilm
  3. 剃刀刀片
  4. 玻璃载玻片(75×38mm)(Thermo Fisher Scientific,Fisher Scientific,目录号:12-550B)
  5. 盖玻片(18×18mm)(厚度:0.17mm)(Thermo Fisher Scientific,Fisher Scientific,目录号:S17521)
  6. 拟南芥野生型(哥伦比亚生态型),dnd1突变体(At5g15410),clv3突变体(At2g27250)
  7. 合成的CLV3肽RTVP p SGP DPLHH 3(GenScript,Piscataway,NJ)
  8. Murashige和Skoog盐(MS)(Caisson Laboratories,目录号:MSP01-10LT)
  9. 氯化钆(III)(GdCl 3)(Sigma-Aldrich,目录号:439770)
  10. 乙醇(Sigma-Aldrich,目录号:459836)
  11. 蔗糖(Sigma-Aldrich,目录号:SO389)
  12. MES缓冲液(pH 5.7)(Caisson Laboratories,目录号:M009-100GM)
  13. 灭菌水
  14. Tris(Sigma-Aldrich,目录号:252859)
  15. 乙酸(Thermo Fisher Scientific,Fisher Scientific,目录号:A38S-500)
  16. 水合氯醛(Sigma-Aldrich,目录号:C8383)
  17. 甘油(Thermo Fisher Scientific,目录号:17904)
  18. 植物培养基(见配方)
  19. 固定解决方案(参见配方)
  20. 清除解决方案(参见配方)

设备

  1. 冰箱
  2. 振动器
  3. 用于生长植物的生长室(100μmolm-2 sec -1白光16小时,黑暗8h,23℃)。
  4. 镊子
  5. 解剖显微镜
  6. 配备有Nomarski光学器件的显微镜(Nikon Instrument,型号:MICROPHOT-FX)。

软件

  1. 无限分析程序(Lumenera,Ottawa,Canada)。

程序

  1. 拟南芥幼苗培养和处理
    1. 向多孔板的每个孔中加入2ml 1/2MS液体培养基。
    2. 对所有种子进行表面消毒,并将1粒种子置于多孔板的每个孔中
    3. 通过将多孔板在4℃放置3天以打破休眠来分层种子。
    4. 分层后,将多孔板从4℃冰箱中取出,并对每个孔添加不同的处理。在我们的实验中,我们添加单独的CLV3配体(1μM终浓度),Ca 2+通道阻断剂GdCl 3单独(150μM终浓度)和CLV3与GdCl 注意:因为CLV3肽溶解在水中,所以对照组应该是水,并且应当是与CLV3处理组相同的量。加入的少量水(在我们的实验中为2μl)不应该对幼苗具有任何渗透胁迫。</em>
    5. 将含有幼苗的多孔板放置在生长室(100μmol/cm 2)中的旋转振荡器(180rpm)上16小时和黑暗在8小时,23℃)。我们将这一天定义为实验的开始日期(第0天)。

  2. 幼苗准备解剖
    1. 在生长室中生长7天后,用镊子从多孔板中一次取出一个幼苗,并将其放在一张纸巾上3秒钟以吸收过量的培养基。
    2. 用镊子从纸巾中取出单个幼苗,并将其侧向放置在解剖显微镜下的一块石蜡膜上。
    3. 用锋利的刀片去除根,子叶和最古老的叶原基。
    4. 为了防止形态变化,快速(但轻轻地)用镊子将切开的组织移动到仅含新鲜制备的固定溶液的同一板的孔中,并在室温下孵育过夜。
    5. 第二天清除固定溶液,更换为90​​%乙醇。将板在室温下振荡器上放置1小时(40rpm)。
    6. 除去90%乙醇,更换为70%乙醇。将板放在振荡器上在室温下长达1小时(40rpm)
    7. 从70%乙醇中一次取出一个切开的组织,并放置在载有一滴新鲜制备的澄清溶液的载玻片上。
    8. 在配有Nomarski光学显微镜(MICROPHOT-FXA;尼康,日本东京)下观察。
      注意:清除溶液的反应是快速(〜5分钟)。最好一次查看1到2个样品,并在显微镜下观察SAM时拍摄照片。一段时间后,SAM的圆顶将难以与周围组织区分开。在显微镜下观察之前,其余的幼苗可以保持在70%乙醇中。

  3. SAM面积测量。
    1. Open Infinity分析软件程序。
    2. 选择"捕获"拍摄照片。在对应于SAM的中间光学截面的焦平面处记录单个幼苗图像
    3. 在两个相对的叶原基的底部边缘之间画一条直线在SAM圆顶的每一边。这条线代表SAM圆顶的底部(图1)。产生垂直线,将SAM基底的中点连接到SAM圆顶的顶部(图1C)。
    4. 选择"区域周长"进行SAM尺寸测量。根据步骤C3中描述的两条线,通过徒手绘制围绕封闭的SAM形状区域。通过计算表示SAM的基底的宽度的直线上方的面积来确定SAM的面积。程序将自动计算面积和周长。
       

      图1.装备有Nomarski光学的显微镜下的7-d-old拟南芥 SAM区域。 A.野生型。 B缺失突变体缺失功能性环核苷酸门控阳离子通道同种型2(CNGC2)多肽和(C)clv3突变体。黄色三角形表示SAM圆顶的基边。连接2个黄色三角形的红线表示SAM圆顶的底部。垂直红线将SAM基座的中点连接到SAM圆顶的顶部。

数据分析

为了比较野生型,dnd1 和 clv3 突变体幼苗的SAM大小(参见图1和图2A),测量了每种基因型的至少15个幼苗。对于野生型中的内源性CLV3(图2B)和外源CLV3对clv3突变体的影响的钙通道阻断剂效应,对每种处理测量至少10个秧苗(图2C)。使用ANOVA分析来评估平均值分离。对于图2A和2B,在代表基因型或治疗的条上的星号或两个星号表示SAM大小显着不同(在P <0.05或 P <0.01) ,分别)比对照(WT)。对于图2C,ANOVA比较用括号表示

图2. Ca 2 + 信号与SAM区域的CLV3控制相互作用。(a)。 7-d-old WT,dnd1 和 clv3 幼苗的SAM区。 (b)。在第3天或第0天(实验开始的当天)在标准1/2MS液体培养基(对照)或补充有Gd 3+ + /μM的培养基上生长的WT幼苗。 (C)。用水(对照),CLV3或CLV3和Gd 3+处理在标准培养基上生长的幼苗。 CLV3和Gd <3> 。使用ANOVA分析评估平均值分离。图2所示的所有幼苗是7日龄幼苗。对于(a)和(b),表示基因型或处理的条上方的星号或两个星号表明SAM大小显着不同(在P <0.05或< < 0.01)。对于(c),ANOVA比较用括号表示。

食谱

  1. 植物培养基
    在200ml MiliQ水中溶解2g蔗糖,0.433g 1/2 MS盐和2ml MES,然后用Tris调节pH至5.7。高压灭菌器
  2. 固定解决方案
    乙酸:无水乙醇(1:9,v/v)
  3. 清除解决方案
    水合氯醛,甘油和水(8:1:2,w/v/v)

致谢

这项工作是由国家科学基金会奖1146827(到G.A.B)支持。该方案从Fiers等人(2006),Ohyama等人(2009),Carles等人改编和修改。 ,(2010)和Miguel Aguilar博士。

参考文献

  1. Aguilar M. 识别突变种子的特征使用nomarski显微镜(基因一)
  2. Carles,C.C.,Ha,C.M.,Jun,J.H.,Fiume,E。和Fletcher。 JC(2010)。  分析茎尖分生组织发育。/a> Plant Development Biol 655:105-29。
  3. Fiers,M.,Golemiec,E.,Schors,R.,Van,Der。,Geest,L.,Van,D.,Li,KW,Stiekema,WJand Liu,CM(2006)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/16751438"target ="_ blank"> CLAVATA3的CLAVATA3/ESR基序在功能上与非保守侧翼序列无关。/a> Plant Physiol 141(4):1284-1292
  4. Ohyama,K.,Shinohara,H.,Ogawa-Ohnishi,M.and Matsubayashi,Y。(2009)。  拟南芥中的糖肽调节干细胞命运。 Natal Biol Biol 5(8):578 -580。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chou, H., Wang, H. and Berkowitz, G. A. (2016). Shoot Apical Meristem Size Measurement. Bio-protocol 6(23): e2055. DOI: 10.21769/BioProtoc.2055.
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