Heterochronic Phenotype Analysis of Hypodermal Seam Cells in Caenorhabditis elegans
秀丽隐杆线虫下皮干细胞的异时表型分析   

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Molecular Cell
Mar 2018

 

Abstract

Heterochrony refers to changes in the timing of developmental events, and it is precisely regulated in the organisms by the heterochronic genes such as C. elegans lin-4 and let-7. Mutations in these genes cause precocious or retarded development of certain cell lineages. With well-defined cell lineages, C. elegans is one of the best model systems to study heterochronic genes, since the subtle changes in the development of cell lineages can be easily identified. Among the different cell types in C. elegans, hypodermal seam cells and their lineages are well known to be maintained by lin-14, whose expression level is regulated by two miRNA genes, lin-4 and let-7, at the larval stages. Therefore, analyzing the heterochronic phenotype of hypodermal seam cells in C. elegans could yield detailed insights into the status of the miRNA pathway. Here we describe the assay protocol to analyze the heterochronic phenotypes of C. elegans hypodermal seam cells, which can be used as a reliable method to study the miRNA pathway.

Keywords: C. elegans (秀丽隐杆线虫), Hypodermal seam cells (下皮干细胞), Heterochronic phenotype (异时表型), miRNA (miRNA), lin-4 (lin-4), let-7 (let-7), lin-14 (lin-14)

Background

Caenorhabditis elegans is a transparent nematode which is found in the soil. It was introduced as a new model organism by Sydney Brenner in the 1960s to study neural development. Since then, it has been extensively studied because of the simple anatomy, the easy cultivation, and the rapid growth. The reproductive life cycle of C. elegans, which takes only three days, consists of the embryonic stage, four larval stages (L1-L4), and the adult stage. After 14 h of embryogenesis, C. elegans grows in size during the four larval stages which are divided by each molt, then it reaches the adult stage which can produce the next generation. In unfavorable conditions, C. elegans larvae can choose the alternative developmental pathway, so-called “dauer”, which can survive a few months in the adverse conditions. When the environment becomes favorable, C. elegans exits the dauer stage and develops into the L4 stage.

In C. elegans, most hypodermal seam cells have the characteristic of stem cells. In each molt, they divide into two daughter cells; the anterior daughter cell fuses with hyp7, which is the major hypodermis, and the posterior daughter cell continues to divide like the stem cells until they terminally differentiate at the L4 stages. Thus, ten hypodermal seam cells at L1 stage generate sixteen hypodermal seam cells at the end of the L4 stage in each side (Figure 1) (Altun and Hall, 2009).

Functionally, hypodermal seam cells are important for the formation of stage-specific cuticles that are composed of various collagen proteins (Thein et al., 2003). They also produce cuticular alae, which are the protruding ridges extending longitudinally along the two sides of the animal over the seam cells (Singh and Sulston, 1978). Alae are produced only in the L1 stage, dauer larva, and adult. Therefore, adult alae are commonly used as an indicator which shows terminal differentiation of hypodermal seam cells. For example, lin-4 or let-7 loss-of-function mutant, which shows the retarded development of hypodermal seam cells, has the alae defects in adult stages, while lin-14 loss-of-function mutant, which shows the precocious development of hypodermal seam cells, has alae in the L3 stage (Hong et al., 2000).

The cell fate of a hypodermal seam cell is regulated by lin-14, whose expression is high in L1 animals, and decreases by the L2 stage (Ruvkun and Giusto, 1989). The lin-14 expression is regulated by well-characterized miRNAs lin-4 and let-7 (Ambros, 1989; Reinhart et al., 2000). It is known that lin-4 and let-7 bind the 3’ untranslated region of lin-14 and downregulate its expression at the larval stages (Lee et al., 1993, Slack et al., 2000). When the expression level of lin-14 is not decreased in the larval stages, hypodermal seam cells abnormally proliferate, generating retarded phenotypes in lin-14 gain-of-function mutants and lin-4 loss-of-function mutants.


Figure 1. Hypodermal seam cell lineage in C. elegans. A. Ten hypodermal seam cells at the L1 stage. These cells generate major hypodermis hyp7, hypodermal seam cells, neurons and glia throughout the development. B. Cell division patterns of hypodermal seam cells in each molt. Most of the hypodermal seam cells generate one terminally differentiated daughter cell and one stem-cell-like seam cell in each division. By the L4 stage, sixteen of hypodermal seam cells are generated in each lateral side.

LIN-14 is a transcription factor that regulates its target gene expressions. One of the target genes of LIN-14 transcription factor is a cell cycle inhibitor cki-1. Inactivation of cki-1. results in the division of the vulva precursor cell (VPC) during the L2 stage. Therefore, decreasing lin-14 expression via lin-4 miRNA is important for the development of vulva as well as the hypodermal seam cell.

miRNAs are small, non-coding RNAs that are produced by a series of RNA-processing steps from their precursors known as pri-miRNAs. The pri-miRNAs are generated by transcription via RNA polymerase II (Bartel, 2004; Lee et al., 2004) and then cleaved by the RNase III endonuclease Drosha to produce pre-miRNAs in the nucleus (Bracht et al., 2004; Lee et al., 2002). These pre-miRNAs are then transported into the cytoplasm by Exportin-5 (Yi et al., 2003). In the cytoplasm, the pre-miRNA is further processed by Dicer to produce the mature 20-25 nt miRNA (Bernstein et al., 2001; Grishok et al., 2001). The mature miRNA functions as a guide to recruit RNA-induced silencing complex (RISC), which is composed of the miRNA:mRNA duplex, Argonaute, and other proteins (Hammond et al., 2001; Carmell et al., 2002; Caudy et al., 2002; Mourelatos et al., 2002; Caudy et al., 2003). In the RISC complex, Argonaute cleaves the target mRNA when it is activated. In C. elegans, there are about 24 Argonaute proteins (Bartel, 2004; Carmel et al., 2002). Among them, ALG-1 and AGL-2 are appeared to be important for downregulating of lin-4 and let-7 targets, because alg-1 and alg-2 mutants share the phenotypes with lin-4 and let-7 mutants. (Grishok et al., 2001).

Here we describe the assay protocols to analyze the heterochronic phenotypes of hypodermal seam cells in C. elegans. These assays are useful for the analysis of the miRNA pathway by taking advantage of the fact that hypodermal seam cell fate is regulated by the well-characterized miRNAs, lin-4 and let-7 (Zhang et al., 2018).

Materials and Reagents

  1. 60 mm Petri dishes (Fisher Scientific, FisherbrandTM, catalog number: AS4051)
  2. 100 mm Petri dishes (Fisher Scientific, FisherbrandTM, catalog number: FB0875712)
  3. Syringe Filter Unit, 0.22 μm (Millipore Sigma, Millex®-GV, catalog number: SLGV033RS)
  4. Frosted microscope slides 25 x 75 x 1.0 mm (Fisher Scientific, FisherbrandTM, catalog number: 12-552-3)
  5. Microscopic cover glass 18 x 18 mm (Fisher Scientific, FisherbrandTM, catalog number: 12-542A)
  6. C. elegans wild type: N2 Bristol strain from C. elegans Genetic Center
  7. JR672 wIs54 [Pscm::gfp] V; wIs54 is the integration allele of the seam cell-specific transcriptional GFP reporter that is expressed in all of the seam cells at all developmental stages (Terns et al., 1997; Koh and Rothman, 2001)
  8. E. coli OP50-1: streptomycin resistant strain from C. elegans Genetic Center
  9. Levamisole (Sigma-Aldrich, catalog number: L9756)
  10. NaCl (Fisher Scientific, Fisherbrand, catalog number: S271, CAS 7647-14-5)
  11. Agar, Bacteriological, Ultrapure (Thermo Scientific, catalog number: J10906, CAS 9002-18-0)
  12. Peptone (BD Bioscience, BD BactoTM, catalog number: 211677)
  13. Calcium Chloride hexahydrate (Sigma-Aldrich, catalog number: 442909)
  14. Magnesium sulfate (Sigma-Aldrich, catalog number: 208094)
  15. Potassium phosphate dibasic (Sigma-Aldrich, catalog number: P3786)
  16. Potassium phosphate monobasic (Sigma-Aldrich, catalog number: P0662)
  17. Cholesterol (Sigma-Aldrich, catalog number: C75209)
  18. Pure alcohol 200 proof (Pharmco products, catalog number: 111000200)
  19. Tryptone (BD Bioscience, BD BactoTM, catalog number: 211705)
  20. Yeast Extract (BD Bioscience, BD BactoTM, catalog number: 212750)
  21. Nail polish
  22. Nematode Growth Media (NGM) Agar (see Recipes)
  23. 1 M CaCl2 (see Recipes)
  24. 0.5 M MgSO4 (see Recipes)
  25. 1 M Potassium phosphate (pH 6) (see Recipes)
  26. 5 mg/ml cholesterol (see Recipes)
  27. LB agar (see Recipes)
  28. LB (see Recipes)
  29. OP50-1 culture (see Recipes)

Equipment

  1. Amsco® Century SV-120 Scientific Prevacuum Sterilizer (STERIS)
  2. 4 L flask
  3. Stir bar
  4. Stir plate
  5. PourBoy® 4 Sterile Media Dispenser (TritechTM Research)
  6. Home-made cell spreader (made with glass Pasteur pipets by bent by heating, spreader size is less than 1 inch.)
  7. 37 °C incubator (VWR, model: 1535)
  8. Incubator Shaker (Eppendorf, New Brunswick Scientific, model: I2500, catalog number: M1284-0000)
  9. 20 °C incubator for C. elegans culture (Intellus control system) (PERCIVAL, model: I-36NL)
  10. A home-made worm picker with a 5¾ glass Pasteur pipet (Fisher Scientific, FisherbrandTM, catalog number: 13-678-6A) and a platinum wire (Scientific Instrument Services, Inc., catalog number: W414)
  11. Fluorescent Stereo Microscope (Leica Microsystems, model: Leica M165FC) with Leica PLANAPO 2.0x objective lens
  12. Confocal laser scanning microscopy platform (Leica Microsystems, model: Leica TCS SP8) with Leica CTR6500 electronics box

Software

  1. GraphPad Prism 7.0a (GraphPad Software, Inc., www.graphpad.com
  2. Leica Application Suite Advanced Fluorescence (Leica Microsystems)

Part I: Analysis of vulva phenotypes

Procedure

  1. Synchronize the populations and grow the worms (Figure 2A)
    1. Transfer twenty one-day-old adult worms, which have approximately ten eggs in the uterus, onto 60 mm NGM plates seeded with E. coli (OP50-1).
      Note: If your mutant of interest has smaller brood size or egg-laying defects, you may need to transfer more worms onto each plate. 
    2. Make 5 replicates of plates per strain.
    3. Let them lay eggs for two hours. More than fifty eggs per plate are needed. If you have less than fifty eggs per plate, go back to Step A1 and increase the number of adult worms in each plate.
    4. Remove the adult worms.
    5. Let the offspring grow until they reach the L4 stage (~48 h) at 20 °C. The L4 stage can be distinguished by a small white crescent spot in the vulva area as well as the size of the worms (Fielenbach and Antebi, 2008).

  2. Analyzing the vulva phenotypes (Figure 2B)


    Figure 2. The flow chart of vulva phenotype analysis. A. Synchronization of the worm population. Eggs are collected from young adult worms for two hours, then they are cultured for approximately 48 h until they reach the L4 stage. B. Analysis of vulva phenotype. Count the total number of worms on Day 0. Red X indicates the worms that show vulva phenotype. Count the worms that show the vulva defects and remove them to avoid repeated counting on following days.

    1. Count the total number of L4 worms as soon as they reach the L4 stage. Typically, each plate has approximately one hundred worms.
    2. After 24 h, censor and remove the worms that show protruded vulva or bagging. Continue with counting and censoring for 3 days.
    3. Calculate the percentages of vulva defects (Zhang et al., 2018).

Data analysis

  1. More than 250 worms should be analyzed for each strain, and three independent experiments should be performed.
  2. Calculate the percentage of worms having vulva defects using the following equation:



  3. Enter the value into GraphPad Prism and analyze the mean with SEM and P-value with two-tailed Student’s t-test.

Part II: Analysis of Alae Phenotypes (Figure 3)

Procedure

  1. Synchronize the worms as described above.
  2. Transfer either young adult or the L3 animals to a new plate. Place ten animals per plate. Make five replicates per strain. 
  3. Analyze the alae phenotype. Under the stereomicroscope, focus on the upper side of each worm under the 24x objective lens. You will see the protruding ridge of the alae on the upper side of the worm from the head to the tail. Observe the alae and record whether they are intact, discontinued, or absent in each individual animal. Refer to Slack et al., 2000 for the alae phenotypes. In the adult stage, discontinued or absent alae are abnormal, while the presence of alae is abnormal at the L3 stage.
  4. Count the number of worms that have intact, discontinued, or absent alae (Zhang et al., 2018).


    Figure 3. The flow chart of alae phenotype analysis. When synchronized worms reach the specific stage, ten worms are transferred into the new plate and the alae phenotypes are analyzed under the microscope.

Data analysis

  1. For each strain, at least 50 worms are observed and three independent experiments are performed.
  2. Calculate the percentage of worms having abnormal alae.
  3. Means with SEM and P-value with two-tailed Student’s t-test are calculated using GraphPad Prism.

Part III: Analysis of the number of hypodermal seam cells

Procedure

  1. Generating the desired strains expressing Pscm::gfp using JR672 (Figure 4)


    Figure 4. Schematic diagrams of genetic crosses for Pscm::gfp expressing mutants. JR672 is crossed with wild-type males to get the GFP-expressing heterozygote males, which are mated with mutants of interests on Day 4. Then, the GFP-expressing hermaphrodites in the F2 generation are the double heterozygotes. With the Mendelian Segregation, the double heterozygotes from the F2 generation would give rise to double homozygotes with a 1/16 chance, when two mutations are not linked. However, since we select only the GFP-expressing worms, the chance for the double homozygotes is increased to 1/12. The green boxes in F3 indicate the possible genotypes of GFP-expressing worms and the red box indicates the genotype of the double mutant.

    1. Prepare NGM plates for the crosses by seeding 20 μl of OP50 in the middle of 60 mm plates. By making a small bacterial lawn, the chances of worm mating are increased.
    2. Cross hermaphrodite of the strain JR672 with N2 males. Hermaphrodite to male ratio for the crosses is usually 1:3. 
    3. On Day 3, remove the parent worms to avoid mixing of two generations.
    4. On Day 4, select F1 males, which are GFP-positive heterozygote to set up a cross with hermaphrodites of your mutant of interest. Keep the hermaphrodite to male ratio 1:3.
    5. On Day 6, remove the parent worms from the plate. Now, it is expected to have only the F2 generation as larvae or eggs in the plate.
    6. On Day 7, select the hermaphrodites that express GFP in the hypodermal seam cells. There is a 50% chance to get the GFP-expressing hermaphrodites if the mating is successful. Clone out a couple of GFP-expressing hermaphrodites, one animal per seeded plate.
    7. On Day 9, remove the parent worm from the plate.
    8. On Day 10, chose one plate which has a good number of worms. Discard the rest of the plates. From the chosen plate, single out GFP-expressing worms. The chance to get the double mutant is one out of twelve if the mutation is not in the same linkage group as the GFP locus, because only the GFP-expressing worms are selected for analysis. Based on the chance to getting double mutants, select the appropriate number of F3 worms. To increase probability of getting double mutants, pick three to four times number of worms. We usually select forty worms if the mutation of interest is not linked with Pscm::gfp. Note that Pscm::gfp is located on linkage group V.
    9. On Day 12, genotype F3 worms for the mutation of interest and select the plates which have homozygotes. Check the plates that have homozygous mutants and identify the ones with all F4 animals positive for GFP expression. If desired, GFP homozygotes can be selected by PCR in F3 generation.

  2. Synchronizing and preparing the worms for microscopy
    1. To obtain a synchronized population of animals, allow ten to twenty of fertile adult worms to lay eggs for two hours and let them grow until they reach the L4 stages at 20 °C as described in Part I Procedure A.
    2. Prepare the 2% agarose pads on the imaging slide as described before (Huynh et al., 2018).
    3. Place ten L4 worms in 7 μl of 10 mM levamisole on the 2% agarose pad, put a coverslip and stabilize the coverslip by painting four corners of coverslip with transparent nail polish.
    4. Under the 40x objective lens, check the GFP signals. GFP is expressed in the nuclei of hypodermal seam cells. Wild-type worms have sixteen hypodermal seam cells on each side at L4 stage (Figure 5), thus the total number of hypodermal seam cells per worm is 32. By focusing up and down, count the number of hypodermal seam cells on each side. In the tail, the focal planes of right and left hypodermal seam cells are closer than others. Thus it is easier to count the total number of hypodermal seam cells by counting one side of cells from the head to tail and the other side from tail to head.


      Figure 5. GFP-expression pattern of Pscm::gfp in wild-type C. elegans. A. GFP is expressed in the nuclei of hypodermal seam cells. A 40x objective lens was used in this image. B. Sixteen hypodermal seam cells are shown in wild-type C. elegans under the 10x objective lens. The smear GFP signal is from the other side of hypodermal seam cells (Zhang et al., 2018).

Data analysis

  1. Analyze at least 30 worms per strain to count the seam cells, and three independent experiments are performed.
  2. Count the total numbers of hypodermal seam cells per worm and calculate the average, SEM, and P-value with two-tailed Student’s t-test using GraphPad Prism.

Notes

  1. The genotype of JR672 is wIs54 [Pscm::gfp] V. Note that Pscm::gfp transgene is located on chromosome V. 
  2. The GFP signal is stronger in the larval stage than in adults. Thus, select the GFP-expressing worm as soon as they reached the L4 stage.

Recipes

  1. Nematode Growth Media (NGM) Agar (2 L)
    1. Add the following ingredients in a 4 L flask:
      NaCl 6.0 g
      Agar, Bacteriological, Ultrapure 34 g
      Bacto peptone 5 g
      dH2O 1,950 ml
      Note: Worms do not grow well on plates prepared with agar of lower grade.
    2. Add a stir bar and mix well. Leave the stir bar inside of the flask
    3. Prepare 1 L of dH2O to wash the tubes for PourBoy® 4 Sterile Media Dispenser, and wrap the tubes with aluminum foil
    4. Autoclave NGM, dH2O and the tubes for PourBoy® 4 Sterile Media Dispenser using liquid 45 min cycle in Amsco® Century SV-120 Scientific Prevacuum Sterilizer
    5. Cool down the NGM on the stir plate until it reaches approximately 55 °C. Meanwhile, prepare 200 of 60 mm Petri-dishes
    6. Once NGM cools down, add the following ingredients:
      2 ml of 1 M CaCl2
      4 ml of 0.5 M MgSO4
      50 ml of Potassium Phosphate (pH 6) and 2 ml Cholesterol (5 mg/ml in ethanol)
    7. Mix well by stirring. It is okay that NGM turns cloudy
    8. Dispense 10 ml of NGM into 60 mm Petri-dish using PourBoy® 4 Sterile Media Dispenser. The NGM plates are dried for a couple of days at RT
    9. Seed OP50-1 as a food source for C. elegans. Add approximately 100 μl of OP50-1 in each plate and spread the bacteria using a home-made cell spreader to make a small circle of bacterial lawn. The bacterial lawn should not touch the edge of the plate. Then, culture the OP50-1 overnight at RT
  2. 1 M CaCl2
    1. Dissolve 21.9 g of Calcium chloride hexahydrate (CaCl2•6H2O) in 90 ml dH2O
    2. Once it is dissolved, add more dH2O to make 100 ml of 1 M CaCl2
    3. Sterilize it by filtering using 0.22 μm filter units
  3. 0.5 M MgSO4
    1. Dissolve 12.03 g of Magnesium sulfate (MgSO4) in 90 ml dH2O
    2. Once it is dissolved, add more dH2O to make 100 ml of 0.5 M MgSO4
    3. Autoclave it with a liquid 45 min cycle in Amsco® Century SV-120 Scientific Prevacuum Sterilizer
  4. 1 M Potassium phosphate (pH 6)
    1. Prepare 100 ml of 1 M K2HPO4 by dissolving 17.42 g of potassium phosphate dibasic in dH2O
    2. Prepare 100 ml of 1 M KH2PO4 by dissolving 13.61 g of potassium phosphate monobasic in dH2O
    3. Take 13.2 ml of 1 M K2HPO4 and add 86.8 ml of 1 M KH2PO4 to make 1 M Potassium phosphate
    4. Autoclave 1 M potassium phosphate with a liquid 45 min cycle in Amsco® Century SV-120 Scientific Prevacuum Sterilizer
  5. 5 mg/ml cholesterol
    Dissolve 0.5 g of cholesterol in 100 ml of ethanol
  6. LB agar
    1. Add the following ingredients:
      NaCl 2.5 g
      Tryptone 2.5 g
      Yeast Extract 1.25 g
      Agar 3.75 g
      dH2O up to 250 ml
    2. Mix well with a stir bar and autoclave with a liquid 45 min cycle
    3. Cool it down to 55 °C and add 250 μl of 50 mg/ml streptomycin
    4. Stir it on the stir plate and pour into 100 mm Petri-dish
    5. Dry the plates on the bench overnight
  7. LB
    1. Add the following ingredients:
      NaCl 10 g
      Tryptone 10 g
      Yeast Extract 5 g
      dH2O up to 1 L
    2. Dissolve by stirring and autoclave with a liquid 45 min cycle
  8. OP50-1 culture
    1. Streak OP50-1 on the LB agar plate containing streptomycin
    2. Culture OP50-1 in 37 °C incubator overnight
    3. Pick one colony of OP50-1 from the LB agar plate and culture in 20 ml of LB containing streptomycin at the final concentration of 50 μg/ml at 37 °C shaking incubator overnight

Acknowledgments

This work was supported by grants from NIH (NS074324, NS089616), The Robert Packard Center for ALS Research at Johns Hopkins, The ALS Association, and The Muscular Dystrophy Association.

Competing interests

The authors have no conflicts of interest or competing interests to declare.

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简介

异时性是指发育事件发生时间的变化,并且通过异时基因如 C在生物体中精确调节。线虫 lin-4 和 let-7 。这些基因的突变导致某些细胞谱系的早熟或延迟发育。有明确定义的细胞谱系, C.线虫是研究异时基因的最佳模型系统之一,因为可以很容易地识别细胞谱系发育的细微变化。在 C中的不同细胞类型中。众所周知,线虫,皮下接缝细胞及其谱系由 lin-14 维持,其表达水平受两个miRNA基因调控, lin-4 和 let-7 ,在幼虫阶段。因此,分析 C中皮下接缝细胞的异时表型。线虫可以提供有关miRNA途径状态的详细见解。在这里,我们描述了分析 C的异时表型的分析方案。线虫皮下接缝细胞,可作为研究miRNA通路的可靠方法。
【背景】 Caenorhabditis elegans 是一种透明的线虫,存在于土壤中。它是由悉尼布伦纳在20世纪60年代引入的一种新的模式生物,用于研究神经发育。从那时起,由于解剖结构简单,易于培养和快速生长,因此进行了广泛的研究。 C的生殖生命周期。仅需三天的线虫由胚胎期,四个幼虫期(L1-L4)和成虫期组成。胚胎发生14小时后, C.线虫在四个幼虫阶段逐渐增大,每个阶段被蜕皮分开,然后到达成虫阶段,可以产生下一代。在不利的条件下, C.线虫幼虫可以选择替代发育途径,即所谓的“dauer”,它可以在不利条件下存活几个月。当环境变得有利时, C.线虫退出dauer阶段并发展到L4阶段。

在 C中。线虫,大多数皮下接缝细胞具有干细胞的特征。在每次蜕皮中,它们分成两个子细胞;前子细胞与hyp7融合,后者是主要的皮下组织,后子细胞继续像干细胞一样分裂,直到它们在L4阶段终末分化。因此,L1阶段的十个皮下接缝细胞在每侧的L4阶段末端产生十六个皮下接缝细胞(图1)(Altun和Hall,2009)。在功能上,皮下接缝细胞对于由各种胶原蛋白组成的阶段特异性角质层的形成是重要的(Thein et al。,2003)。它们还产生角质层,它们是沿着接缝细胞沿动物两侧纵向延伸的突出脊(Singh和Sulston,1978)。 Alae仅在L1阶段,dauer幼虫和成体中产生。因此,成年alae通常用作显示皮下接缝细胞的终末分化的指示剂。例如, lin-4 或 let-7 功能丧失突变体,显示皮下接缝细胞的延迟发育,在成人阶段具有alae缺陷,而 lin-14 功能丧失突变体,其显示皮下接缝细胞的早熟发育,在L3阶段具有alae(Hong et al。,2000)。

皮下接缝细胞的细胞命运受 lin-14 调节,其在L1动物中表达高,并且在L2阶段降低(Ruvkun和Giusto,1989)。 lin-14 表达受到充分表征的miRNA lin-4 和 let-7 的调控(Ambros,1989; Reinhart et al。,2000)。已知 lin-4 和 let-7 结合 lin-14 的3'非翻译区并下调其在幼虫阶段的表达(Lee et al。,1993,Slack et al。,2000)。当 lin-14 的表达水平在幼虫阶段没有减少时,皮下接缝细胞异常增殖,在 lin-14 功能获得突变体中产生延迟的表型,并且 lin-4 功能丧失突变体。


图1. C中的皮下接缝细胞谱系。线虫。 A. L1阶段的十个皮下接缝细胞。这些细胞在整个发育过程中产生主要的皮下组织hyp7,皮下接缝细胞,神经元和神经胶质。 B.每个蜕皮中皮下接缝细胞的细胞分裂模式。大多数皮下接缝细胞在每个分区中产生一个终末分化的子细胞和一个干细胞样接缝细胞。通过L4阶段,在每个侧面产生十六个皮下接缝细胞。

LIN-14是一种调节其靶基因表达的转录因子。 LIN-14转录因子的靶基因之一是细胞周期抑制剂 cki-1。 cki-1的灭活。导致外阴前体细胞的分裂( VPC)在L2阶段。因此,通过 lin-4 miRNA降低 lin-14 表达对于外阴和皮下接缝细胞的发育是重要的。

miRNA是小的非编码RNA,其通过来自其前体(称为pri-miRNA)的一系列RNA加工步骤产生。通过RNA聚合酶II转录产生pri-miRNA(Bartel,2004; Lee et al。,2004),然后通过RNase III内切核酸酶Drosha切割以在细胞核中产生pre-miRNA(Bracht) et al。,2004; Lee et al。,2002)。然后这些pre-miRNA通过Exportin-5转运到细胞质中(Yi et al。,2003)。在细胞质中,pre-miRNA由Dicer进一步处理以产生成熟的20-25nt miRNA(Bernstein et al。,2001; Grishok et al。,2001 )。成熟的miRNA可作为募集RNA诱导的沉默复合物(RISC)的指导,RISC由miRNA:mRNA双链体,Argonaute和其他蛋白质组成(Hammond et al。,2001; Carmell et al。,2002; Caudy et al。,2002; Mourelatos et al。,2002; Caudy et al。 >,2003)。在RISC复合体中,Argonaute在激活时切割靶mRNA。在 C中。线虫,大约有24种Argonaute蛋白(Bartel,2004; Carmel et al。,2002)。其中,ALG-1和AGL-2似乎对 lin-4 和 let-7 目标的下调非常重要,因为 alg-1 和 alg-2 突变体与 lin-4 和 let-7 突变体共享表型。 (Grishok et al。,2001)。

在这里,我们描述了分析 C中皮下接缝细胞的异时表型的分析方案。线虫。这些分析可用于分析miRNA通路,利用皮下接缝细胞命运受到充分表征的miRNA, lin-4 和 let-7 (Zhang et al。,2018)。

关键字:秀丽隐杆线虫, 下皮干细胞, 异时表型, miRNA, lin-4, let-7, lin-14

材料和试剂

  1. 60 mm培养皿(Fisher Scientific,Fisherbrand TM ,目录号:AS4051)
  2. 100毫米培养皿(Fisher Scientific,Fisherbrand TM ,目录号:FB0875712)
  3. 注射器过滤器单元,0.22μm(Millipore Sigma,Millex ® -GV,目录号:SLGV033RS)
  4. 磨砂显微镜载玻片25 x 75 x 1.0 mm(Fisher Scientific,Fisherbrand TM ,目录号:12-552-3)
  5. 微观盖玻片18 x 18 mm(Fisher Scientific,Fisherbrand TM ,目录号:12-542A)
  6. ℃。线虫野生型:来自 C的N2 Bristol菌株。线虫遗传中心
  7. JR672 wIs54 [P scm :: gfp ] V; wIs54 是接缝细胞特异性转录GFP报告基因的整合等位基因,在所有发育阶段的所有接合细胞中均有表达(Terns et al。,1997; Koh和Rothman,2001)
  8. 电子。大肠杆菌OP50-1:来自 C的链霉素抗性菌株。线虫遗传中心
  9. 左旋咪唑(Sigma-Aldrich,目录号:L9756)
  10. NaCl(Fisher Scientific,Fisherbrand,目录号:S271,CAS 7647-14-5)
  11. 琼脂,细菌,超纯(Thermo Scientific,目录号:J10906,CAS 9002-18-0)
  12. 蛋白胨(BD Bioscience,BD Bacto TM ,目录号:211677)
  13. 六水合氯化钙(Sigma-Aldrich,目录号:442909)
  14. 硫酸镁(Sigma-Aldrich,目录号:208094)
  15. 磷酸氢二钾(Sigma-Aldrich,目录号:P3786)
  16. 磷酸二氢钾(Sigma-Aldrich,目录号:P0662)
  17. 胆固醇(Sigma-Aldrich,目录号:C75209)
  18. 纯酒精200证明(Pharmco产品,目录号:111000200)
  19. 胰蛋白胨(BD Bioscience,BD Bacto TM ,目录号:211705)
  20. 酵母提取物(BD Bioscience,BD Bacto TM ,目录号:212750)
  21. 指甲油
  22. 线虫生长培养基(NGM)琼脂(见食谱)
  23. 1 M CaCl 2 (见食谱)
  24. 0.5 M MgSO 4 (见食谱)
  25. 1 M磷酸钾(pH 6)(见食谱)
  26. 5毫克/毫升胆固醇(见食谱)
  27. LB琼脂(见食谱)
  28. LB(见食谱)
  29. OP50-1文化(见食谱)

设备

  1. Amsco ®世纪SV-120科学预真空灭菌器(STERIS)
  2. 4升烧瓶
  3. 搅拌棒
  4. 搅拌盘
  5. PourBoy ® 4无菌培养基分配器(Tritech TM 研究)
  6. 自制细胞分布器(玻璃巴斯德吸管通过加热弯曲,扩展器尺寸小于1英寸。)
  7. 37°C培养箱(VWR,型号:1535)
  8. Incubator Shaker(Eppendorf,New Brunswick Scientific,型号:I2500,目录号:M1284-0000)
  9. 用于 C的20°C培养箱。线虫文化(Intellus控制系统)(PERCIVAL,型号:I-36NL)
  10. 自制的蠕虫捡拾器,带有5¾玻璃巴斯德吸管(Fisher Scientific,Fisherbrand TM ,目录号:13-678-6A)和铂丝(Scientific Instrument Services,Inc。,目录号: W414)
  11. 徕卡PLANAPO 2.0x物镜的荧光立体显微镜(徕卡显微系统,型号:Leica M165FC)
  12. 共聚焦激光扫描显微镜平台(徕卡显微系统,型号:Leica TCS SP8)配Leica CTR6500电子盒

软件

  1. GraphPad Prism 7.0a(GraphPad Software,Inc., www.graphpad.com ) 
  2. Leica Application Suite高级荧光(徕卡显微系统)

第一部分:外阴表型分析

程序

  1. 同步种群并种植蠕虫(图2A)
    1. 将20只一日龄成虫(在子宫中大约有10个卵子)转移到接种 E的60mm NGM平板上。大肠杆菌(OP50-1)。
      注意:如果您感兴趣的突变体具有较小的育雏大小或产卵缺陷,您可能需要将更多的蠕虫转移到每个平板上。 
    2. 每个菌株制备5个重复的平板。
    3. 让他们产卵两个小时。每个板需要超过五十个鸡蛋。如果每个板的蛋数少于50个,请返回步骤A1并增加每个板中成虫的数量。
    4. 去除成虫。
    5. 让后代生长,直到它们在20℃达到L4阶段(~48小时)。 L4阶段可以通过外阴区域的小白色新月点以及蠕虫的大小来区分(Fielenbach和Antebi,2008)。

  2. 分析外阴表型(图2B)


    图2.外阴表型分析的流程图。 :一种。蠕虫群体的同步。从年轻成虫中收集鸡蛋两小时,然后将它们培养约48小时直至它们达到L4阶段。 B.外阴表型分析。计算第0天的蠕虫总数。红色X表示显示外阴表型的蠕虫。计算显示外阴缺陷的蠕虫并将其移除以避免在接下来的几天重复计数。

    1. 一旦到达L4阶段,就计算L4蠕虫的总数。通常,每个板具有大约一百个蠕虫。
    2. 24小时后,检查并移除显示突出的外阴或套袋的蠕虫。继续计数和审查3天。
    3. 计算外阴缺陷的百分比(Zhang et al。,2018)。

数据分析

  1. 每种菌株应分析250多种蠕虫,并应进行三次独立实验。
  2. 使用以下等式计算具有外阴缺陷的蠕虫的百分比:



  3. 在GraphPad Prism中输入值,用SEM和 P -value分析平均值,用双尾学生 t - 测试。

第二部分:Alae表型分析(图3)

程序

  1. 如上所述同步蠕虫。
  2. 将年轻成人或L3动物转移到新的平板上。每板放置10只动物。每个菌株重复5次。 
  3. 分析alae表型。在立体显微镜下,将焦点放在24x物镜下的每个蜗杆的上侧。你会看到从头到尾的虫子上侧的突出的脊。观察alae并记录每只动物的完整,停止或缺失。有关alae表型,请参阅Slack et al。,2000。在成人阶段,停止或不存在alae是异常的,而在L3阶段alae的存在是异常的。
  4. 计算完整,已停止或缺少alae的蠕虫数量(Zhang et al。,2018)。


    图3. alae表型分析的流程图。 当同步的蠕虫到达特定阶段时,将10个蠕虫转移到新的平板中,并在显微镜下分析alae表型。

数据分析

  1. 对于每个菌株,观察到至少50个蠕虫并且进行三个独立的实验。
  2. 计算有异常的蠕虫的百分比。
  3. 使用GraphPad Prism计算具有双尾学生 t - 测试的SEM和 P 值的均值。

第三部分:皮下接缝细胞数量的分析

程序

  1. 使用JR672生成表达P scm :: gfp 的所需菌株(图4)


    图4. P scm :: gfp 表达突变体的遗传杂交示意图。 JR672与野生杂交获得表达GFP的杂合子雄性的雄性,其在第4天与感兴趣的突变体交配。然后,F2代中表达GFP的雌雄同体是双杂合子。使用孟德尔分离,来自F2代的双杂合子将产生双纯合子,当两个突变没有连锁时,具有1/16的机会。然而,由于我们只选择表达GFP的蠕虫,因此双纯合子的机会增加到1/12。 F3中的绿色框表示表达GFP的蠕虫的可能基因型,红色框表示双突变体的基因型。

    1. 通过在60 mm平板的中间接种20μlOP50来准备用于杂交的NGM板。通过制作小的细菌草坪,蠕虫交配的机会增加。
    2. 将菌株JR672的雌雄同体与N2雄性杂交。雌雄同体与雄性的比例通常为1:3。 
    3. 在第3天,移除父蠕虫以避免混合两代。
    4. 在第4天,选择F1雄性,它们是GFP阳性杂合子,与您感兴趣的突变体的雌雄同体建立杂交。保持雌雄同体与雄性比例为1:3。
    5. 在第6天,从盘子中移除父蠕虫。现在,预计只有F2代作为幼虫或盘中的蛋。
    6. 在第7天,选择在皮下接缝细胞中表达GFP的雌雄同体。如果交配成功,有50%的机会获得表达GFP的雌雄同体。克隆了几个表达GFP的雌雄同体,每个种子板一只动物。
    7. 在第9天,从板上移除母虫。
    8. 在第10天,选择一个具有大量蠕虫的平板。丢弃其余的盘子。从选择的平板中,挑出表达GFP的蠕虫。如果突变不在与GFP基因座相同的连锁群中,则获得双突变体的机会是十二分之一,因为仅选择表达GFP的蠕虫进行分析。基于获得双突变体的机会,选择适当数量的F3蠕虫。为了增加获得双突变体的可能性,选择三到四倍的蠕虫数量。如果感兴趣的突变与P scm :: gfp 无关,我们通常会选择40种蠕虫。请注意,P scm :: gfp 位于连锁组V.
    9. 在第12天,为感兴趣的突变基因型F3蠕虫并选择具有纯合子的平板。检查具有纯合突变体的平板,并鉴定具有GFP表达阳性的所有F4动物的平板。如果需要,可以在F3代中通过PCR选择GFP纯合子。

  2. 同步和准备用于显微镜的蠕虫
    1. 为了获得同步的动物群体,允许10到20只可繁殖的成虫在2小时产卵并让它们生长,直到它们达到20℃的L4阶段,如第I部分程序A所述。
    2. 如前所述,在成像载玻片上制备2%琼脂糖垫(Huynh et al。,2018)。
    3. 将10个L4蠕虫置于2%琼脂糖垫上的7μl10mM左旋咪唑中,盖上盖玻片并通过用透明指甲油涂覆盖玻片的四个角来稳定盖玻片。
    4. 在40倍物镜下,检查GFP信号。 GFP在皮下接缝细胞的细胞核中表达。野生型蠕虫在L4阶段每侧有16个皮下接缝细胞(图5),因此每个蠕虫的皮下接缝细胞总数为32.通过上下聚焦,计算每侧皮下接缝细胞的数量。在尾部,左右皮下接缝细胞的焦平面比其他细胞更接近。因此,通过计算从头到尾和从尾到头的另一侧细胞的一侧,可以更容易地计算皮下接缝细胞的总数。


      图5.野生型 scm :: gfp 的GFP表达模式 ℃。线虫 。 A. GFP在皮下接缝细胞的细胞核中表达。在该图像中使用40倍物镜。 B.在野生型 C中显示了十六个皮下接缝细胞。 10x物镜下的线虫。涂片GFP信号来自皮下接缝细胞的另一侧(Zhang et al。,2018)。

数据分析

  1. 分析每个菌株至少30个蠕虫来计算接缝细胞,并进行三次独立实验。
  2. 计算每个蠕虫的皮下接缝细胞总数,并使用GraphPad Prism计算双尾学生 t - 测试的平均值,SEM和 P 值。

笔记

  1. JR672的基因型是 wIs54 [P scm :: gfp ] V.注意P scm :: gfp 转基因位于染色体V上。  
  2. GFP信号在幼虫期比在成年期更强。因此,一旦它们到达L4阶段就选择表达GFP的蠕虫。

食谱

  1. 线虫生长培养基(NGM)琼脂(2升)
    1. 在4升烧瓶中加入以下成分:
      NaCl 6.0克
      琼脂,细菌,超纯34克
      Bacto蛋白胨5克
      dH 2 O 1,950 ml
      注意:在用低等级琼脂制备的平板上,蠕虫不能很好地生长。
    2. 加入搅拌棒搅拌均匀。将搅拌棒留在烧瓶内
    3. 准备1升dH 2 O清洗PourBoy ® 4无菌介质分配器的管子,并用铝箔包裹管子
    4. 高压灭菌NGM,dH 2 O和用于PourBoy ® 4无菌介质分配器的管在Amsco ® Century SV-120 Scientific中使用液体45分钟循环预真空灭菌器
    5. 冷却搅拌盘上的NGM,直至达到约55°C。同时,准备200个60毫米培养皿
    6. 一旦NGM冷却,添加以下成分:
      2毫升1M CaCl 2
      4毫升0.5M MgSO 4
      50毫升磷酸钾(pH 6)和2毫升胆固醇(5毫克/毫升乙醇)
    7. 搅拌均匀。 NGM变得多云是可以的
    8. 使用PourBoy ® 4无菌培养基分配器将10 ml NGM分配到60 mm培养皿中。将NGM板在室温下干燥几天
    9. 种子OP50-1作为 C的食物来源。线虫。在每个平板中加入约100μl的OP50-1,并使用自制的细胞分布器扩散细菌,制成一小圈细菌草坪。细菌草坪不应接触板的边缘。然后,在室温下培养OP50-1过夜
  2. 1 M CaCl 2
    1. 将21.9克氯化钙六水合物(CaCl 2 •6H 2 O)溶于90毫升dH 2 O中
    2. 一旦溶解,加入更多dH 2 O,制成100 ml的1M CaCl 2
    3. 使用0.22μm过滤装置过滤灭菌
  3. 0.5 M MgSO 4
    1. 将12.03克硫酸镁(MgSO 4 )溶于90毫升dH 2 O中
    2. 一旦溶解,加入更多dH 2 O,制成100 ml 0.5 M MgSO 4
    3. 在Amsco ® Century SV-120 Scientific Prevacuum Sterilizer中以45分钟的液体循环高压灭菌
  4. 1 M磷酸钾(pH 6)
    1. 通过将17.42g磷酸氢二钾溶解在dH 2 O中制备100ml的1M K 2 HPO 4
    2. 通过将13.61g磷酸二氢钾溶解在dH 2 O中制备100ml的1M KH 2 PO 4
    3. 取13.2ml 1MK 2 HPO 4 并加入86.8ml 1M KH 2 PO 4 制成1 M磷酸钾
    4. 在Amsco ® Century SV-120 Scientific Prevacuum Sterilizer中高压灭菌1M磷酸钾,液体循环45分钟
  5. 5毫克/毫升胆固醇
    将0.5克胆固醇溶于100毫升乙醇中
  6. LB琼脂
    1. 添加以下成分:
      NaCl 2.5克
      胰蛋白胨2.5克
      酵母提取物1.25克
      琼脂3.75克
      dH 2 O至250毫升
    2. 用搅拌棒和高压釜充分混合,液体循环45分钟
    3. 将其冷却至55℃并加入250μl50mg/ ml链霉素
    4. 将其在搅拌板上搅拌并倒入100mm培养皿中
    5. 将板在板凳上干燥过夜
  7. LB
    1. 添加以下成分:
      NaCl 10克
      胰蛋白胨10克
      酵母提取物5克
      dH 2 O至1升
    2. 通过搅拌和高压釜溶解45分钟的液体
  8. OP50-1文化
    1. 在含有链霉素的LB琼脂平板上划线OP50-1
    2. 在37°C培养箱中培养OP50-1过夜
    3. 从LB琼脂平板上挑取一个OP50-1菌落,在含有50μg/ ml终浓度的链霉素的LB中于37℃振荡培养箱中培养过夜。

致谢

这项工作得到了NIH(NS074324,NS089616),约翰霍普金斯大学ALS研究的Robert Packard中心,ALS协会和肌肉营养不良协会的资助。

利益争夺

作者没有利益冲突或竞争利益申报。

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引用:Ji, Y. J. and Wang, J. (2019). Heterochronic Phenotype Analysis of Hypodermal Seam Cells in Caenorhabditis elegans. Bio-protocol 9(1): e3132. DOI: 10.21769/BioProtoc.3132.
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