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Validating Candidate Congenital Heart Disease Genes in Drosophila
果蝇中候选先天性心脏病基因的验证   

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Abstract

Genomic sequencing efforts can implicate large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system to validate candidate gene association with pathology is therefore useful. We present such a system employing Drosophila to validate candidate congenital heart disease (CHD) genes. The protocols exploit comprehensive libraries of UAS-GeneX-RNAi fly strains that when crossed into a 4XHand-Gal4 genetic background afford highly efficient cardiac-specific knockdown of endogenous fly orthologs of human genes. A panel of quantitative assays evaluates phenotypic severity across multiple cardiac parameters. These include developmental lethality, larva and adult heart morphology, and adult longevity. These protocols were recently used to evaluate more than 100 candidate CHD genes implicated by patient whole-exome sequencing (Zhu et al., 2017).

Keywords: Drosophila(果蝇), High-throughput screening(高通量筛选), Congenital heart disease(先天性心脏病), Lethal rate(致死率), Heart morphology(心脏形态)

Background

The use of the Drosophila model to elucidate molecular mechanisms underlying human diseases is well documented (Bier and Bodmer, 2004; Cagan, 2011; Zhang et al., 2013; Owusu-Ansah and Perrimon, 2014; Diop and Bodmer, 2015; Na et al., 2015), and 75% of human disease associated genes are represented by functional homologs in the fly genome (Reiter et al., 2001). While it is a challenge to link Drosophila developmental phenotypes directly to patient symptoms, Drosophila can be used as a very sophisticated and efficient platform to test and validate candidate disease gene function in development, and this can readily be scaled to evaluate a large number of candidate genes identified from patient genomic sequencing efforts. Drosophila has been used to study genes related to CHD for over 20 years, based on evolutionarily conserved genetic mechanisms of heart development (Bier and Bodmer, 2004; Olson, 2006; Yi et al., 2006). We developed a highly efficient cardiac-targeted gene silencing approach in flies to examine effects on heart structure and function for fly homologs of candidate CHD genes (Zhu et al., 2017).

Materials and Reagents

  1. 1,250 μl pipette tips (BioExpress, GeneMate, catalog number: P-1234-1250 )
  2. 200 μl pipette tips (BioExpress, GeneMate, catalog number: P-1237-200 )
  3. 10 μl pipette tips (BioExpress, GeneMate, catalog number: P-1234-10XL )
  4. Permanent marker
  5. FisherbrandTM plastic Petri dishes (Fisher Scientific, catalog number: S33580A )
  6. Microscope slides (VWR, catalog number: 16004-430 )
  7. 24 x 50 mm gold SealTM cover slips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3322 )
  8. Heart-specific Gal4 driver line, 4XHand-Gal4/Cyo (Generated by Dr. Zhe Han)
  9. UAS RNAi transgenic strains targeting Drosophila orthologs of candidate CHD genes (Bloomington Drosophila Stock Center)
  10. Carbon dioxide (Roberts Oxygen Company)
  11. Fly food (Meidi Laboratories)
  12. Vaseline (COVIDIENTM)
  13. Schneider’s Drosophila medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21720001 )
  14. Paraformaldehyde solution, 4% in PBS (Alfa Aesar, Affymetrix/USB, catalog number: J19943 )
  15. Phosphate buffered saline (PBS), prepared from 10x PBS, pH 7.4 (GENAXY, catalog number: 40-029 )
  16. Bovine serum albumin, Powder (Santa Cruz Biotechnology, catalog number: sc-2323 )
  17. Triton X-100 (Fisher Scientific, catalog number: BP151-100 )
  18. Alexa Fluor 555 Phalloidin (Thermo Fisher Scientific, InvitrogenTM, catalog number: A34055 )
  19. EC11 anti-Pericardin primary anti-mouse antibody (Developmental Studies Hybridoma Bank, catalog number: EC11 )
  20. Biotin-conjugated goat anti-mouse antibody (Vector Laboratories, catalog number: SP-1100 )
  21. Streptavidin (Cy5) (Thermo Fisher Scientific, InvitrogenTM, catalog number: SA1011 )
  22. VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, catalog number: H-1200 )
  23. Electron microscopy science clear nail polish (Electron Microscopy Science, catalog number: 72180 )

Equipment

  1. Gilson P200 pipette classic large plunger (Gilson, model: P200 )
  2. Vannas spring scissors–3 mm cutting Edge (Fine Science Tools, catalog number: 15000-10 )
  3. Dumont #5 forceps (Roboz Surgical Instrument, catalog number: RS-4955 )
  4. Ultimate Flypad (Genesee Scientific, catalog number: 59-172 )
  5. Stereo microscope (ZEISS, model: Stemi 305 )
  6. Zeiss ApoTome.2 microscope using a 20x Plan-Apochromat 0.8 N.A/air objective (ZEISS, model: Apotome.2 )
  7. Drosophila incubator set to 25 °C and 29 °C (Panasonic Healthcare, model: MIR-154-PA )

Software

  1. ImageJ software Version 1.49

Procedure

  1. High-throughput gene function validation system in Drosophila
    1. 5 male flies homozygous for a UAS-RNAi transgene (targeting the Drosophila ortholog of candidate CHD gene) are combined with 10 to 15 4-day-old virgin female flies of genotype 4XHand-Gal4/Cyo (Figure 1) at 25 °C.


      Figure 1. 5 male homozygous UAS-RNAi transgenic flies are crossed to 10-15 4-day-old virgin female flies of genotype 4XHand-Gal4/Cyo

    2. The flies are maintained at 25 °C and are transferred daily to a fresh vial of fly food for 5-6 days.
    3. Each day the emptied vial containing freshly laid eggs is transferred to 29 °C to boost UAS-transgene expression. At the end of this process, 5-6 vials of flies are developing at 29 °C.
    4. As adult progeny flies emerge over a period of four to five days, they are anesthetized with CO2 on a fly pad (Genesee Scientific) and the numbers of curly winged (CyO, no RNAi transgene) vs. straight winged (RNAi transgene expressed in cardioblasts) flies are recorded (Figure 2). Counting continues until at least 200 curly winged flies have been recorded.


      Figure 2. Progeny adult flies emerge and the number of adult flies with curly wings (CyO, no transgene) vs. straight wings (RNAi transgene expressed in cardioblasts) are recorded. An example of lethal rate of approx. 65% is shown.

    5. The [developmental] lethal rate attributable to target gene silencing in the heart is calculated as (Curly - Straight)/Curly x 100% = % Mortality.

  2. Adult survival assay
    1. At least 60 adult progeny flies with straight wings (4XHand-Gal4 driven UAS-RNAi transgene expression in cardioblasts) are collected and maintained at 29 °C. This number of flies can typically be collected in one to two days. Maintain no more than 15 flies per vial. To obtain sufficient numbers of straight winged progeny flies at least 5 crosses should be set up. In the case of RNAi transgenes that induce high mortality, more than 10 crosses should be established.
    2. The survival assay initiates immediately upon fly collection. The number of live flies is thereafter recorded every two days until all flies have died.
    3. Flies are transferred every two days to a fresh vial of fly food (to prevent flies from becoming stuck in wet food).
    4. A survival curve is generated that plots % surviving flies against time (Figure 3).


      Figure 3. Survival curves for control flies (no RNAi transgene) and flies expressing RNAi targeting the Rbbp5 gene in cardioblasts. Heart-specific Rbbp5 knockdown significantly reduces longevity.

  3. Adult heart morphology
    1. Six to ten straight wing adult progeny flies (RNAi transgene expressed in cardioblasts) and six to ten curly wing control progeny adult flies (no RNAi transgene) are anesthetized with CO2 and carefully immobilized (ventral side up) in the bottom of a Petri dish by gently affixing flies in vaseline (Vogler and Ocorr, 2009). Each fly genotype is inscribed directly on the petri dish using a permanent marker. The flies remain in the same Petri dish throughout the procedure until being mounted for microscopic examination. Processing experimental and control flies simultaneously eliminates variability that might result from separate treatments.
    2. The legs are removed by amputation using fine spring scissors.
    3. Schneider’s Drosophila medium (~20 ml per dish) is added by pouring from one side of the Petri dish until flies are completely submerged.
    4. Using scissors, begin at the rostral end of the fly and cut circumferentially and continuously to remove the entire ventral abdominal cuticle, revealing the inner organs.
    5. Remove the internal organs (viscera) that tend to float free of the abdominal cavity by pipetting adjacent medium up and down 5 to 6 times using a P200 pipette set at 200 μl volume.
    6. Remnant viscera and fat body tissue are delicately cleared away using fine forceps (Figure 4).


      Figure 4. Adult fly dissection. Step 1: Stick the fly on the Petri dish; Step 2: Remove the fly legs; Step 3: Open the body from bottom; Step 4: Remove the organs.

    7. Pour out the original medium. Add fresh Schneider’s Drosophila medium (~20 ml) to the Petri dish by pouring from one side until flies are completely submerged to wash the fly carcasses. Pour out the wash medium.
    8. Add ~20 ml formaldehyde solution (4% in PBS) to the Petri dish by pouring from one side until flies are completely submerged. Fix for 10 min at room temperature. Pour out the fixative solution.
    9. Rinse carcasses by adding ~20 ml PBS to the Petri dish by pouring from one side until flies are completely submerged. Pour out the PBS. Repeat the rinse procedure another 2 x.
    10. Add ~20 ml BSA solution (2% in PBS) containing 0.1% Triton X-100 to the Petri dish by pouring from one side until flies are completely submerged. Incubate for 30 min at room temperature. Pour out the BSA solution.
    11. Add ~20 ml PBS containing Alexa Fluor 555 Phalloidin (1:1,000 dilution) and anti-Pericardin (EC11) mouse primary antibody (1:500 dilution) to the Petri dish by pouring from one side until flies are completely submerged. Incubate overnight at 4 °C in the dark.
    12. Remove Petri dish from 4 °C.
    Note: The following steps need not be performed in the dark.
    1. Rinse 3 x with PBS at room temperature as described in step C9.
    2. Add ~20 ml PBS and incubate for 20 min at room temperature. Repeat 3 x.
    3. Add ~20 ml PBS containing Biotin-conjugated goat anti-mouse antibody (1:500 dilution). Incubate for 2 h at room temperature.
    4. Rinse 3 x with PBS at room temperature as described in step C9.
    5. Add ~20 ml PBS and incubate for 20 min at room temperature. Repeat 3 x.
    6. Add ~20 ml PBS containing Streptavidin Cy5 (1:1,000 dilution) and incubated for 1 h at room temperature.
    7. Rinse 3 x with PBS at room temperature as described in step C9.
    8. Add ~20 ml PBS and incubate for 20 min at room temperature. Repeat 3 x.
    9. Mount heart tissue on a glass slide in ~2 ml VECTASHIELD antifade mounting medium.
    10. Apply a cover slip using forceps.
    11. Seal edges of cover slip using nail polish.
    12. Confocal imaging is performed using a Zeiss ApoTome.2 microscope fitted with a 20x Plan-Apochromat 0.8 N.A/air objective.
    13. Control groups are imaged first to establish light intensity and exposure time. An exposure time is found at which the image is saturated, and then reduced to a set point of approx. 70% saturation to allow comparison of fluorescence intensity across genotypes.
    14. The entire heart is imaged by collecting Z-stack images. Same number of samples of each phenotype are imaged.
    15. Images are exported to tiff file format.
    16. ImageJ software Version 1.49 is used for image processing.
    17. Z-stack projections are screened and image levels containing cardiac myofibers are selected for analysis, avoiding the ventral muscle layer that underlies the heart tube (Figure 5).
    18. Samples of reduced myofibrillar density and increased pericardin deposition are shown (Figure 6).


      Figure 5. Z-stack layers that exclude ventral muscle fibers are selected for analysis. Scare bar = 50 μm.


      Figure 6. Samples of reduced myofibrillar density and increased pericardin deposition. Scale bar = 50 μm.

  4. 3rd instar larva heart morphology
    1. Larvae are grown at 29 °C to the 3rd instar stage. Six to ten control larvae (lacking RNAi transgene) are selected on the basis of GFP expression in the head, detected by fluorescence stereo microscopy (the GFP marker is linked to CyO on the inherited balancer chromosome). Six to ten experimental larvae carrying a UAS-RNAi transgene expressed in cardioblasts are identified by the absence of GFP marker expression (Brent et al., 2009).
    2. Using forceps insert insect pins into tail and head to affix larvae to Petri dish, ventral side UP.
    3. Submerge each larva under a drop of Schneider’s Drosophila medium.
    4. Using scissors, make a ventral incision through the cuticle from tail to head.
    5. Insert another 4 insect pins to hold open the excised cuticle.
    6. The internal organs are carefully removed using No. 5 forceps (Figure 7).


      Figure 7. Dissection process of 3rd instar larva. Step 1: Put the larva on the Petri dish; Step 2: Use insect pins to secure the larva; Step 3: Open the body from bottom to top; Step 4: Secure the open cuticle with another 4 insect pins; Step 5: Remove the organs. Scale bar ≈ 0.25 mm.

    7. Wash the remaining larva carcass 1 x with Schneider’s Drosophila medium.
    8. The remaining steps are identical to the adult protocol.

Data analysis

  1. Use Freehand selection of ImageJ to carefully select the same area of all tissue samples.
  2. Cardiac myofibrillar density, cardioblast cell numbers, and Pericardin deposition are quantified.
  3. Use PAST.exe to perform statistical analysis. Sample error is presented as standard error of the mean (SEM). First test results for normality using the Shapiro-Wilk test (a = 0.05). Analyze normally distributed data by Student’s t-test (two groups) and Bonferroni comparison to adjust P value, or by a one-way analysis of variance followed by a Tukey-Kramer post-test for comparing multiple groups. Analyze non-normally distributed data by either a Mann-Whitney test (two groups) and Bonferroni comparison to adjust the P value, or a Kruskal-Wallis H-test followed by a Dunn’s test for comparisons between multiple groups. Statistical significance is defined as P < 0.05.

Notes

  1. All samples are imaged 1x only to avoid bleaching. All samples are imaged the same day at the same light intensity and exposure time.
  2. No ventral muscle layer is present at 3rd instar larval stage.

Acknowledgments

Z.H. was supported by grants from the National Institutes of Health (RO1-HL090801, RO1-NK098410).

References

  1. Bier, E. and Bodmer, R. (2004). Drosophila, an emerging model for cardiac disease. Gene 342(1): 1-11.
  2. Brent, J. R., Werner, K. M. and McCabe, B. D. (2009). Drosophila larval NMJ dissection. J Vis Exp 24: 1107.
  3. Cagan, R. L. (2011). The Drosophila nephrocyte. Curr Opin Nephrol Hypertens 20(4): 409-415.
  4. Diop, S. B. and Bodmer, R. (2015). Gaining insights into diabetic cardiomyopathy from Drosophila. Trends Endocrinol Metab 26(11): 618-627.
  5. Na, J., Sweetwyne, M. T., Park, A. S., Susztak, K. and Cagan, R. L. (2015). Diet-induced podocyte dysfunction in Drosophila and mammals. Cell Rep 12(4): 636-647.
  6. Olson, E. N. (2006). Gene regulatory networks in the evolution and development of the heart. Science 313(5795): 1922-1927.
  7. Owusu-Ansah, E. and Perrimon, N. (2014). Modeling metabolic homeostasis and nutrient sensing in Drosophila: implications for aging and metabolic diseases. Dis Model Mech 7(3): 343-350.
  8. Reiter, L. T., Potocki, L., Chien, S., Gribskov, M. and Bier, E. (2001). A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster. Genome Res 11(6): 1114-1125.
  9. Vogler, G. and Ocorr, K. (2009). Visualizing the beating heart in Drosophila. J Vis Exp (31).
  10. Yi, P., Han, Z., Li, X. and Olson, E. N. (2006). The mevalonate pathway controls heart formation in Drosophila by isoprenylation of Gγ1. Science 313(5791): 1301-1303.
  11. Zhang, F., Zhao, Y., Chao, Y., Muir, K. and Han, Z. (2013). Cubilin and amnionless mediate protein reabsorption in Drosophila nephrocytes. J Am Soc Nephrol 24(2): 209-216.
  12. Zhu, J. Y., Fu, Y., Nettleton, M., Richman, A. and Han, Z. (2017). High throughput in vivo functional validation of candidate congenital heart disease genes in Drosophila. Elife 6.

简介

基因组测序工作可能涉及大量基因和从头突变作为潜在的疾病风险因素。 因此,用于验证候选基因与病理学关联的高通量体内模型系统是有用的。 我们提出了使用果蝇来验证候选先天性心脏病(CHD)基因的这种系统。 这些方案利用了UAS-GeneX-RNAi飞行菌株的综合文库,当跨越4XHand-Gal4遗传背景时,提供了人类基因内源性直向同源物的高效心脏特异性敲低。 一组定量测定评估多种心脏参数的表型严重程度。 这些包括发育致死率,幼虫和成人心脏形态以及成年人的长寿。 这些方案最近被用于评估患者全外显子测序所涉及的100多个候选CHD基因(Zhu等,2017)。
【背景】(Bier和Bodmer,2004; Cagan,2011; Zhang et al。,2013; Owusu-Ansah和Perrimon,2014; Diop和Bodmer,2015; Na et al。等等,2015),75%的人类疾病相关基因由飞行基因组中的功能同系物表示(Reiter et al。,2001)。尽管将果蝇发育表型直接与患者症状联系起来是一个挑战,果蝇可以作为一个非常复杂和有效的平台来测试和验证候选疾病基因功能在开发中,这可以很容易地扩大到评估大量的候选基因从患者基因组测序工作中确定。基于进化保守的心脏发育遗传机制,果蝇已被用于研究与冠心病相关20多年的基因(Bier和Bodmer,2004; Olson,2006; Yi et al。,2006)。我们开发了一种高效的心脏靶向基因沉默法,用于检查候选CHD基因的蝇类同源物对心脏结构和功能的影响(Zhu et al。,2017)。

关键字:果蝇, 高通量筛选, 先天性心脏病, 致死率, 心脏形态

材料和试剂

  1. 1,250μl移液器吸头(BioExpress,GeneMate,目录号:P-1234-1250)
  2. 200μl移液器吸头(BioExpress,GeneMate,目录号:P-1237-200)
  3. 10μl移液管吸头(BioExpress,GeneMate,目录号:P-1234-10XL)
  4. 永久性标记
  5. Fisherbrand TM 塑料培养皿(Fisher Scientific,目录号:S33580A)
  6. 显微镜幻灯片(VWR,目录号:16004-430)
  7. 24 x 50毫米金密封 TM 盖板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3322)
  8. 心脏特异性Gal4驱动线,4XHand-Gal4/Cyo(由Zhe Han博士生成)
  9. 靶向果蝇的UAS RNAi转基因株系候选CHD基因的直向同源物(Bloomington果蝇库存中心)
  10. 二氧化碳(Roberts Oxygen Company)
  11. 飞食(Meidi Laboratories)
  12. 凡士林(COVIDIEN TM
  13. 施耐德果蝇培养基(Thermo Fisher Scientific,Gibco TM ,目录号:21720001)
  14. PBS中的4%的Paraformaldehyde溶液(Alfa Aesar,Affymetrix/USB,目录号:J19943)
  15. 磷酸盐缓冲盐水(PBS),由10倍PBS,pH 7.4(GENAXY,目录号:40-029)制备
  16. 牛血清白蛋白,粉末(Santa Cruz Biotechnology,目录号:sc-2323)
  17. Triton X-100(Fisher Scientific,目录号:BP151-100)
  18. Alexa Fluor 555鬼笔环肽(Thermo Fisher Scientific,Invitrogen TM,目录号:A34055)
  19. EC11抗心包蛋白原抗体抗体(Developmental Studies Hybridoma Bank,目录号:EC11)
  20. 生物素缀合的山羊抗小鼠抗体(Vector Laboratories,目录号:SP-1100)
  21. 链霉亲和素(Cy5)(Thermo Fisher Scientific,Invitrogen TM,目录号:SA1011)
  22. VECTASHIELD带有DAPI的抗变形载体(Vector Laboratories,目录号:H-1200)
  23. 电子显微镜科学清除指甲油(电子显微镜科学,目录号:72180)

设备

  1. Gilson P200移液器经典大柱塞(Gilson,型号:P200)
  2. 万纳斯弹簧剪3毫米切削刃(精细科学工具,目录号:15000-10)
  3. Dumont#5镊子(Roboz Surgical Instrument,目录号:RS-4955)
  4. Ultimate Flypad(Genesee Scientific,目录号:59-172)
  5. 立体显微镜(ZEISS,型号:Stemi 305)
  6. Zeiss ApoTome.2显微镜,使用20x Plan-Apochromat 0.8 N.A/air objective(ZEISS,型号:Apotome.2)
  7. 果蝇孵化器设置为25°C和29°C(Panasonic Healthcare,型号:MIR-154-PA)

软件

  1. ImageJ软件版本1.49

程序

  1. 果蝇中的高通量基因功能验证系统
    1. 与UAS-RNAi转基因(靶向候选人CHD基因的果蝇直系同源物)的5只雄性苍蝇与10至15个4日龄的基因型4XHand-Gal4/Cyo的雌性雌性动物(图1)在25°C

      图1. 5只雄性纯合UAS-RNAi转基因苍蝇与基因型4XHand-Gal4/Cyo的10-15只4日龄的未知女性苍蝇交叉

    2. 苍蝇保持在25°C,并每天转移到新鲜的一小瓶飞行食物5-6天
    3. 每天将含有新鲜产卵的清空小瓶转移到29℃以促进UAS-转基因表达。在该过程结束时,在29℃下发展出5-6个小瓶的苍蝇。
    4. 随着成年后代飞行四至五天的时间,它们在飞垫(Genesee Scientific)上用CO 2进行麻醉,卷曲(CyO,无RNAi转基因)与直线记录有翅(RNAi转基因在成体细胞中表达)苍蝇(图2)。计数持续到至少有200只卷曲的苍蝇被记录下来

      图2.记录了后代的成年苍蝇,并记录了具有卷曲翅膀(CyO,无转基因)的成年苍蝇与直翅膀(在成体细胞中表达的RNAi转基因)的数量。致死率约为。 65%显示。

    5. 归因于心脏中靶基因沉默的[发育]致死率计算为(Curly-Straight)/Curly×100%=%死亡率。

  2. 成人生存分析
    1. 收集至少60只具有直翅膀的成年后代(4XHand-Gal4驱动的UAS-RNAi转基因在成核细胞中的表达)并保持在29℃。这个数量的苍蝇通常可以在一到两天内收集。每瓶维持不超过15只苍蝇。要获得足够数量的直翅膀后代,至少要有5个十字架。在引起高死亡率的RNAi转基因的情况下,应建立十多个杂交
    2. 飞行收集后立即开始生存测定。此后每两天记录一次活蝇数,直到所有苍蝇死亡为止。
    3. 每两天将苍蝇转移到一个新鲜的小食物上(以防止苍蝇被卡在湿食物中)。
    4. 产生生存曲线,绘制百分比幸存的苍蝇与时间(图3)

      图3.对照蝇(无RNAi转基因)和表达靶向成核细胞中Rbbp5基因的RNAi的苍蝇的存活曲线。心脏特异性Rbbp5敲低显着减少寿命。

  3. 成人心脏形态
    1. 将6至10只直翅成虫后代(在成体细胞中表达的RNAi转基因)和6至10个卷曲翼控制后代成虫(无RNAi转基因)用CO 2麻醉并仔细固定(腹侧向上)通过轻轻地将苍蝇粘在凡士林中,在培养皿的底部(Vogler和Ocorr,2009)。每只蝇基因型使用永久标记直接刻在培养皿上。苍蝇在整个过程中保持在同一培养皿中,直到被安装用于显微镜检查。处理实验和对照蝇同时消除可能由单独处理引起的变异性。
    2. 使用细弹簧剪刀将腿部截肢。
    3. 通过从培养皿的一侧倾倒直到苍蝇被完全浸没,加入施奈德氏果蝇培养基(〜20ml /碟)。
    4. 使用剪刀,从飞行的末端开始,沿圆周方向连续切开以去除整个腹侧腹部角质层,露出内脏。
    5. 通过使用设定为200μl体积的P200移液管将相邻培养基上下移液5至6次,从而去除倾向于游离腹腔的内脏(内脏)。
    6. 残余的内脏和脂肪的身体组织用细镊子精心清除(图4)

      图4.成人飞行解剖。步骤1:将飞行物贴在培养皿上;步骤2:去除飞腿;步骤3:从底部打开身体;步骤4:去除器官。

    7. 倾出原来的媒介。通过从一侧倾倒将新鲜的施奈德的果蝇培养基(〜20毫升)添加到培养皿中,直到苍蝇完全浸没以洗涤飞行尸体。倒出洗涤介质。
    8. 通过从一侧倾注直到苍蝇完全浸没,将〜20 ml甲醛溶液(4%在PBS中)添加到培养皿中。在室温下固化10分钟。倾倒固定液。
    9. 通过从一侧倾倒直到苍蝇完全浸没,向培养皿中加入〜20毫升PBS冲洗屠体。倒出PBS。再次冲洗2 x。
    10. 将含有0.1%Triton X-100的〜20ml BSA溶液(2%在PBS中)从一侧倒入直至苍蝇完全浸没。在室温下孵育30分钟。倒出BSA溶液。
    11. 通过从一侧倾倒将含有Alexa Fluor 555 Phalloidin(1:1,000稀释)和抗Pericardin(EC11)小鼠原代抗体(1:500稀释))的约20 ml PBS加入到培养皿中,直到苍蝇完全浸没。在黑暗中4℃孵育过夜。
    12. 从4°C去除培养皿。
    注意:以下步骤不需要在黑暗中执行。
    1. 在室温下用PBS冲洗3次,如步骤C9所述。
    2. 加入〜20 ml PBS,室温孵育20 min。重复3 x。
    3. 加入含有生物素缀合的山羊抗小鼠抗体(1:500稀释)的〜20ml PBS。在室温下孵育2小时。
    4. 在室温下用PBS冲洗3次,如步骤C9所述。
    5. 加入〜20 ml PBS,室温孵育20 min。重复3 x。
    6. 加入含有链霉亲和素Cy5(1:1,000稀释液)的〜20ml PBS,室温孵育1 h。
    7. 在室温下用PBS冲洗3次,如步骤C9所述。
    8. 加入〜20 ml PBS,室温孵育20 min。重复3 x。
    9. 将载玻片上的心脏组织装入〜2 ml VECTASHIELD防霉剂安装介质。
    10. 用镊子涂上盖子。
    11. 使用指甲油密封盖子的边缘。
    12. 共聚焦成像使用装有20x Plan-Apochromat 0.8 N.A /空气目标的Zeiss ApoTome.2显微镜进行。
    13. 控制组首先被成像,以建立光强度和曝光时间。发现曝光时间是图像饱和,然后缩小到大约的设定点。 70%的饱和度可以比较基因型的荧光强度
    14. 整个心脏通过收集Z-stack图像成像。每个表型的样本数量相同。
    15. 图像导出为tiff文件格式。
    16. ImageJ软件版本1.49用于图像处理。
    17. 筛选Z-stack投影,并选择含有心肌肌纤维的图像水平进行分析,避免心脏层下面的腹侧肌层(图5)。
    18. 显示减少的肌原纤维密度和增加的心包蛋白沉积的样品(图6)

      图5.选择排除腹侧肌纤维的Z-叠层进行分析。 Scare bar = 50μm。


      图6.减少肌原纤维密度和增加心包蛋白沉积的样品比例尺=50μm。

  4. 3 rd 幼虫心脏形态学
    1. 幼虫在29℃至3℃阶段生长。基于GFP表达,在荧光立体显微镜(GFP标记与遗传平衡染色体上的CyO连接)上检测到6至10个对照幼虫(缺少RNAi转基因)。通过不存在GFP标志物表达(Brent等人,2009)鉴定携带在成核细胞中表达的UAS-RNAi转基因的6至10个实验幼虫。
    2. 使用镊子将昆虫针插入尾巴和头部,将幼虫贴到培养皿,腹侧UP。
    3. 将每只幼虫淹没在一滴施耐德的果蝇介质下。
    4. 使用剪刀,从头到尾通过角质层进行腹侧切口。
    5. 插入另外4个昆虫针,以打开切除的角质层。
    6. 使用5号钳子小心取出内脏(图7)

      图7. 3℃的解剖过程 instar幼虫。步骤1:将幼虫置于培养皿上;步骤2:使用昆虫针固定幼虫;步骤3:从下到上打开身体;步骤4:用另外4个昆虫针固定开放的角质层;步骤5:取出器官。比例尺≈0.25 mm

    7. 用施耐德的果蝇培养基清洗剩余的幼虫尸体1号。
    8. 其余步骤与成人方案相同。

数据分析

  1. 使用ImageJ的Freehand选择,仔细选择所有组织样本的相同区域。
  2. 量化心肌肌原纤维密度,成纤维细胞数和心包蛋白沉积
  3. 使用PAST.exe执行统计分析。样品误差以平均值(SEM)的标准误差表示。使用Shapiro-Wilk检验的第一次检测结果(a = 0.05)。通过Student's t -test(两组)和Bonferroni比较来分析正态分布的数据,以调整P 值,或通过单向方差分析后跟Tukey-Kramer后测试比较多组。通过Mann-Whitney测试(两组)和Bonferroni比较来分析非正态分布的数据,以调整 P 值,或者使用Kruskal-Wallis H检验,然后进行Dunn测试,以进行多重比较组。统计学意义被定义为 P 0.05。

笔记

  1. 所有样品均为1x成像,以避免漂白。所有样品在相同的光强度和曝光时间的同一天进行成像。
  2. 在3日龄幼虫期不存在腹肌层

致谢

Z.H.得到了国家卫生研究院(RO1-HL090801,RO1-NK098410)的资助。

参考

  1. Bier,E.和Bodmer,R。(2004)。  果蝇,一种新兴的心脏病模型。 基因 342(1):1-11。
  2. Brent,JR,Werner,KM和McCabe,BD(2009)。  果蝇幼虫NMJ解剖。 J Vis Exp 24:1107.
  3. Cagan,RL(2011)。果蝇肾细胞。 Curr Opin Nephrol Hypertens 20(4):409-415。
  4. Diop,SB和Bodmer,R。(2015)。从果蝇获得糖尿病性心肌病的见解。趋势内分泌代谢组26(11):618-627。
  5. Na,J.,Sweetwyne,MT,Park,AS,Susztak,K。和Cagan,RL(2015)。
  6. Olson,EN(2006)。基因监管网络进化和发展的心脏。 科学 313(5795):1922-1927。
  7. Owusu-Ansah,E.和Perrimon,N。(2014)。在果蝇中建模代谢体内平衡和营养感觉:对老龄化和代谢疾病的影响 Dis Model Mech 7(3):343-350。 br />
  8. Reiter,LT,Potocki,L.,Chien,S.,Gribskov,M。和Bier,E。(2001)。对果蝇中的人类疾病相关基因序列的系统分析黑腹果蝇。 Genome Res 11(6):1114-1125。
  9. Vogler,G。和Ocorr,K.(2009)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/19786947"target ="_ blank" >可视化果蝇中的跳动心脏 J Vis Exp (31)。
  10. Yi,P.,Han,Z.,Li,X.和Olson,EN(2006)。甲羟戊酸途径通过Gγ1的异戊二酰化控制果蝇中的心脏形成。 313(5791):1301-1303 。
  11. Zhang,F.,Zhao,Y.,Chao,Y.,Muir,K.and Han,Z.(2013)。  Cubilin和无腹膜介导在果蝇中的蛋白质重吸收肾细胞。 J Am Soc Nephrol 24( 2):209-216。
  12. Zhu,JY,Fu,Y.,Nettleton,M.,Richman,A.and Han,Z.(2017)。  体内的高通量功能验证在果蝇中的候选先天性心脏病基因。 > Elife 6.
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Copyright Zhu et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zhu, J., Fu, Y., Richman, A. and Han, Z. (2017). Validating Candidate Congenital Heart Disease Genes in Drosophila. Bio-protocol 7(12): e2350. DOI: 10.21769/BioProtoc.2350.
  2. Zhu, J. Y., Fu, Y., Nettleton, M., Richman, A. and Han, Z. (2017). High throughput in vivo functional validation of candidate congenital heart disease genes in Drosophila. Elife 6.
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