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Chick Neural Tube Explant Culture
鸡胚胎神经管外植体培养   

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Abstract

The neural tube explant culture technique allows in vitro culturing of small pieces of neural tissue isolated from e.g., chick or mouse embryonic tissue in a matrix of collagen for defined periods of time. This method can be used to study the effects of defined molecules on developmental processes such as neural progenitor proliferation and neuronal differentiation and/or survival. Since the explant material can also be prepared from embryonic tissue electroporated with expression vectors, this technique can be adapted to study gene function in the presence of specific environmental signals. Different regions of the neural tube can also be isolated during the dissection step, allowing specific regions of the neural tube to be studied separately. Here, we present a neural tube explant culture method that we have used in several studies (Dias et al., 2014; Lek et al., 2010; Vallstedt et al., 2005).

Materials and Reagents

  1. 10 cm Petri dish (SARSTEDT AG & Co, catalog number: 82.1473 )
  2. Glass Pasteur pipette 150 mm (VWR International, catalog number: 612-1701 )
  3. Transfer pipette (SARSTEDT AG & Co, catalog number: 86.1174 )
  4. Tungsten wire (Goodfellow, catalog number: w005160 )
  5. 10 ml syringe (HARTMANN USA, Omnifix®, catalog number: 4617100V )
  6. Needle 20 G (Henke Sass Wolf, catalog number: 4710009040 )
  7. Fresh fertilized chick eggs (ideally from a local supplier)
  8. L-15 medium (Life Technologies, catalog number: 11415-049 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11415-049”.
  9. Dispase I (Roche Diagnostics, catalog number: 04942086001 )
  10.  Heat inactivated Fetal bovine serum (FBS) ( Life Technologies, GibcoTM, catalog number: 10106-169 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 10106-169 ”.
  11. Dulbecco’s modified eagle’s medium 10x (MEM) (Sigma-Aldrich, catalog number: D2429 )
  12. Sodium hydroxide (NaOH) (EMD Millipore Corporation, catalog number: 1.06469.1000 )
  13. Collagen (3.1 mg/ml) (Pure Col) (Advanced BioMatrix, catalog number: 5005-B )
  14. Sodium bicarbonate 7.5% (BIC) (Life Technologies, catalog number: 25080 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25080”.
  15. DMEM/F-12, GlutaMAXTM supplement (Life Technologies, catalog number: 31331-028 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 31331-028”.
  16. Neurobasal medium (Life Technologies, catalog number: 21103-049 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 21103-049”.
  17. Albumin from bovine serum (BSA) (Sigma-Aldrich, catalog number: A7906 )
  18. Glutamax (Life Technologies, catalog number: 35050-038 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 35050-038”.
  19. Penicillin Streptomycin (PEST) (Life Technologies, catalog number: 15140-122 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122”.
  20. N2-supplement (Life Technologies, catalog number: 17502-048 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 17502-048”.
  21. B27-supplement (Life Technologies, catalog number: 17504-044 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 17504-044”.
  22. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  23. Dispase I solution (see Recipes)
  24. Bedding solution (see Recipes)
  25. Growing media (see Recipes)

Equipment

  1. Humidified, 38 °C egg incubator (Grumbach, catalog number: 8012 )
  2. Humidified, 37 °C tissue culture incubator (Thermo Fisher Scientific, catalog number: 51030287 )
  3. 16 °C incubator (Gemini BV Laboratory,Liebherr, catalog number: 4294 F1-1 )
  4. Dissection microscope and light source (ZEISS, models: Zeiss Stemi SV11 and Zeiss KL1500LCD )
  5. Borosilicate glass capillaries (Harvard Apparatus, catalog number: GC100TF-10 )
  6. Nunc 4-well dishes for IVF (Thermo Fisher Scientific, catalog number: 144444 )
  7. Flaming/brown micropipette puller (SUTTER, catalog number: P-97 )
  8. Needle holder (Fine science tools, catalog number: 26016-12 )
  9. Tweezers (Fine science tools, catalog number: 11252-20 )
  10. Micro-scissors (Fine science tools, catalog number: 15000-00 )
  11. Dissection scissors (Fine science tools, catalog number: 14088-10 )
  12. Mouth aspirator system/Aspirator tube assemblies for calibrated microcapillary pipettes (Sigma-Aldrich, catalog number: A5177-5EA )

Procedure

  1. Isolation of chick embryos
    1. Store fertilized eggs at 16 °C (up to a week) until start of the experiment.
    2. Place eggs horizontally and incubate in a humidified chamber at 38 °C until they reach the desired Hamburger-Hamilton (HH) embryonic stage (Hamburger and Hamilton, 1992). We have used this protocol successfully with chick embryos from embryonic stages HH10-25.
    3. Make a hole in the pole region of the egg and remove approximately 5 ml of albumin (egg white) using a 10 ml syringe-needle to lower down the embryo inside the egg.
    4. Cut an oval window on the top of the egg using dissection scissors.
    5. Remove the embryo from the egg. Use a pair of tweezers to hold the membrane near the embryo and dissection scissors to cut the membrane around the embryo (see Video 1 for steps A3-5). Transfer the embryo with attached membranes into a 10 cm Petri dish on ice containing cold L-15 medium. Remove embryos from all incubated eggs before proceeding to embryo dissection.
    6. Place the Petri dish containing the embryos under a dissection microscope, remove the membranes surrounding the embryos using tweezers and a dissection micro-scissor (Figure 1a-b) and transfer them to new cold L-15 medium on ice with a transfer pipette.
    7. Somite-stage the embryos and keep embryos of the desired developmental stage (Hamburger and Hamilton, 1992).

  2. “Raw” embryo dissection
    Note: From this point onwards, all dissection steps of the embryonic tissue will be performed at RT under dissection microscope.
    1. Place an embryo in a Petri dish containing ice-cold L-15 medium. With a straight tungsten needle keep the embryo immobilized and with a hook-shaped tungsten needle remove as much mesenchyme as possible without damaging the neural tube (see Figure 1b-d).
    2. Cut the region of the neural tube of interest (e.g., Figure 1e) using the hook-shaped needle and transfer the neural tube piece to a four-well plate on ice containing L15 medium using a glass Pasteur pipette.
    3. Repeat steps B1-2 to remaining embryos, one at the time. If suitable, can be pooled to the same well on the four-well plate.

  3. “Fine” embryo dissection
    1. Pull borosilicate glass capillaries using a micropipette puller and cut the tip of the needle with tweezers. Typically, a tip of approximately 0.5 mm in diameter allows an easy manipulation of the dissected tissue (explant).
    2. Using a four-well plate prepare a Dispase I solution well, a 10% FBS/L-15 solution well, and two L-15 medium wells (washing wells). Use a volume of solution sufficient to cover the dissected tissue (500 µl of solution per well is usually sufficient). Keep at room-temperature (RT). Transfer the dissected embryos from step B with a glass Pasteur pipette sequentially through the four wells with an incubation period of 5 min/well at RT. Keep the tissue in the last washing well and place it on ice. Do not incubate the tissue in the Dispase I solution for more than 5 min.
    3. Coat two 10 cm Petri dishes with ~2 ml of FBS and incubate for 2 min at RT. Remove FBS and add ice-cold L-15 medium. Keep dishes on ice.
    4. Transfer one tissue piece to the coated Petri dish and remove the remaining mesenchymal tissue attached to the neural tube using the tungsten needles. Use a straight-shaped needle to hold the tissue and the hook-shaped needle to remove the remaining mesenchyme. Once all mesenchyme is removed the tissue should look “shiny” and smooth (see Figure 1f). Use the hook-shaped tungsten needle to cut the desired region of the neural tube to be cultured (e.g., ventral midline, Figure 1g, intermediate or dorsal region of the neural tube).
    5. Transfer the dissected tissue (explant) with a glass capillary (prepared in step C1) in a mouth aspirator tube to a four-well plate coated with FBS and containing L-15 medium (~600 µl) on ice. Change to a new coated Petri dish after every second tissue dissection to make sure the media in the Petri dish is always cold.

  4. Tissue incubation
    1. Prepare the bedding solution according to Recipes.
    2. Add a 16 µl drop to the bottom of the wells of a four-well plate. Incubate in a humidified tissue culture incubator at 37 °C for 25-30 min, until the bubble becomes slightly “milky” white (Figure 1h, top left well).
    3. Add 2 drops of ~50 µl of bedding solution to the lid of a 10 cm Petri dish. Wash the explants by transferring them with a glass capillary in a mouth aspirator tube from one drop of bedding to the other.
    4. Add ~20 µl of bedding solution on top of the dried bedding drop from step D2 and transfer the washed explants from step D3 to the new drop of bedding. The explant will sink to the top of the dried drop. Position the explant using the tungsten needles (see Figure 1i).
    5. Incubate ~30-60 min in a humidified tissue culture incubator at 37 °C until the bedding drop hardens. Add 650 µl/well of growing medium and incubate at 37 °C. Soluble factors can be added at any time to the growing medium according to experimental setup. Culture the explants for the desired period of time according to the experimental setup.

Representative data

Video 1. Isolation of chick embryos from egg



Figure 1. Representative images of different steps of chick neural tube explant technique. a. Chick embryo isolated from egg; b. Chick embryo isolated from surrounding membranes; c. Chick embryo after “raw” dissection step; d. Tungsten needle holding “raw” dissected embryo; e. Dissected hindbrain region of the neural tube of embryo shown in c; Boxed region in c corresponds to the region shown in e; f. Neural tube after fine dissection of neural tube shown in e. Note that the mesenchyme (M) surrounding the neural tube (NT) in e is now completely removed; g. Isolated ventral region neural tube, corresponding approximately to the boxed region in f; h. 4-well plate containing a bubble of collagen on top left well; i. Bubble of collagen containing 2 isolated explants of the neural tube.

Recipes

  1. Dispase I solution
    Prepare a stock solution by dissolving Dispase I in water to a final concentration of 5 mg/ml
    Aliquot the solution and store at -20 °C up to one year
    Prepare a working solution of Dispase I by diluting the stock solution in L-15 medium to a final concentration of 1 mg/ml
  2. Bedding solution
    Note: This solution should be freshly prepared.
    Add 8.64 µl of 2.5 M NaOH to 200 µl of MEM 10x and vortex
    The solution will turn into a pink color
    Add 1.8 ml of collagen (3.1 mg/ml) and mix
    The solution will turn to an apricot color
    Add 88 µl of BIC and mix
    The final solution should have a pale pink color
    Stored the solution on ice
  3. Growing media
    Mix together 250 ml of DMEM/F12 + Glutamax medium with 250 ml of Neurobasal medium, 2.5 ml of Glutamax, 2.5 ml of N2-supplement, 5 ml of B27-supplemet, 1.3 ml of a BSA stock solution at 10 mg/ml, 5 ml of PEST and 4 µl of 2-mercaptoethanol. Stored at 4 °C up to one month

Acknowledgments

Funding for this work was provided by Cancerfonden and Karolinska Institutet. This protocol was adapted from procedures published in Vallstedt et al. (2005) and Dias et al. (2014).

References

  1. Dias, J. M., Alekseenko, Z., Applequist, J. M. and Ericson, J. (2014). Tgfbeta signaling regulates temporal neurogenesis and potency of neural stem cells in the CNS. Neuron 84(5): 927-939.
  2. Hamburger, V. and Hamilton, H. L. (1992). A series of normal stages in the development of the chick embryo. 1951. Dev Dyn 195(4): 231-272.
  3. Lek, M., Dias, J. M., Marklund, U., Uhde, C. W., Kurdija, S., Lei, Q., Sussel, L., Rubenstein, J. L., Matise, M. P., Arnold, H. H., Jessell, T. M. and Ericson, J. (2010). A homeodomain feedback circuit underlies step-function interpretation of a Shh morphogen gradient during ventral neural patterning. Development 137(23): 4051-4060.
  4. Vallstedt, A., Klos, J. M. and Ericson, J. (2005). Multiple dorsoventral origins of oligodendrocyte generation in the spinal cord and hindbrain. Neuron 45(1): 55-67.

简介

神经管外植体培养技术允许在胶原基质中从例如小鸡或小鼠胚胎组织分离的小片神经组织的体外培养定义的时间段。 该方法可用于研究限定分子对发育过程如神经祖细胞增殖和神经元分化和/或存活的影响。 由于外植体材料也可以从用表达载体电穿孔的胚胎组织制备,该技术可以适于在特定环境信号的存在下研究基因功能。 也可以在解剖步骤期间分离神经管的不同区域,允许单独研究神经管的特定区域。 在这里,我们提出了我们已经在几个研究中使用的神经管外植体培养方法(Dias等人,2014; Lek等人,2010; Vallstedt, et al。,2005)。

材料和试剂

  1. 10cm培养皿(SARSTEDT AG& Co,目录号:82.1473)
  2. 玻璃巴斯德移液管150mm(VWR International,目录号:612-1701)
  3. 转移移液管(SARSTEDT AG& Co,目录号:86.1174)
  4. 钨丝(Goodfellow,目录号:w005160)
  5. 10ml注射器(HARTMANN USA,Omnifix ,目录号:4617100V)
  6. 针20G(Henke Sass Wolf,目录号:4710009040)
  7. 新鲜受精鸡蛋(最好来自当地供应商)
  8. L-15培养基(Life Technologies,目录号:11415-049) 注意:目前,它是"Thermo Fisher Scientific,Gibco TM ,目录号:11415-049"。
  9. Dispase I(Roche Diagnostics,目录号:04942086001)
  10. &热灭活胎牛血清(FBS)(Life Technologies,Gibco TM ,目录号:10106-169)
    注意:目前,它是"Thermo Fisher Scientific,Gibco TM,目录号:10106-169"。
  11. Dulbecco's改良的Eagle培养基10x(MEM)(Sigma-Aldrich,目录号:D2429)
  12. 氢氧化钠(NaOH)(EMD Millipore Corporation,目录号:1.06469.1000)
  13. 胶原(3.1mg/ml)(Pure Col)(Advanced BioMatrix,目录号:5005-B)
  14. 碳酸氢钠7.5%(BIC)(Life Technologies,目录号:25080)
    注意:目前,是"Thermo Fisher Scientific,Gibco TM ,目录号:25080" />
  15. DMEM/F-12,GlutaMAX 补充剂(Life Technologies,目录号:31331-028)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:31331-028"
  16. Neurobasal培养基(Life Technologies,目录号:21103-049)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:21103-049" 。
  17. 来自牛血清(BSA)的白蛋白(Sigma-Aldrich,目录号:A7906)
  18. Glutamax(Life Technologies,目录号:35050-038)
    注意:目前,是"Thermo Fisher Scientific,Gibco TM ,目录号:35050-038"
  19. 青霉素链霉素(PEST)(Life Technologies,目录号:15140-122)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:15140-122"
  20. N2补充剂(Life Technologies,目录号:17502-048)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:17502-048"
  21. B27-补充剂(Life Technologies,目录号:17504-044)
    注意:目前,是"Thermo Fisher Scientific,Gibco TM ,目录号:17504-044"
  22. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  23. Dispase I解决方案(参见配方)
  24. 寝具解决方案(见配方)
  25. 生长介质(见配方)

设备

  1. 加湿,38℃鸡蛋培养箱(Grumbach,目录号:8012)
  2. 加湿,37℃组织培养箱(Thermo Fisher Scientific,目录号:51030287)
  3. 16℃培养箱(Gemini BV Laboratory,Liebherr,目录号:4294 F1-1)
  4. 解剖显微镜和光源(ZEISS,型号:Zeiss Stemi SV11和Zeiss KL1500LCD)
  5. 硼硅酸盐玻璃毛细管(Harvard Apparatus,目录号:GC100TF-10)
  6. 用于IVF(Thermo Fisher Scientific,目录号:144444)的Nunc 4孔培养皿
  7. 火焰/棕色微量移液器(SUTTER,目录号:P-97)
  8. 针架(Fine science tools,目录号:26016-12)
  9. 镊子(精细科学工具,目录号:11252-20)
  10. 微型剪刀(Fine science tools,目录号:15000-00)
  11. 解剖剪刀(精细科学工具,目录号:14088-10)
  12. 用于校准的微毛细管移液管(Sigma-Aldrich,目录号:A5177-5EA)的抽吸器系统/抽吸管组件

程序

  1. 隔离雏鸡胚胎
    1. 在16℃(长达一周)存储受精卵,直到实验开始。
    2. 水平放置鸡蛋,在38℃的加湿室中孵育 ℃,直到它们达到期望的汉堡 - 汉密尔顿(HH)胚胎阶段 (Hamburger和Hamilton,1992)。我们已成功使用此协议 与胚胎阶段HH10-25的鸡胚。
    3. 打个洞 鸡蛋的极区,并去除大约5ml的白蛋白(鸡蛋 ?白色),使用10毫升注射器针降低胚胎内 ?蛋。
    4. 使用解剖剪刀在蛋顶部切割一个椭圆形窗口。
    5. 从鸡蛋中取出胚胎。使用一对镊子握住 膜附近的胚胎和解剖剪刀切膜 (参见视频1的步骤A3-5)。转移胚胎 ?将膜连接到含有冷L-15的冰上的10cm陪替氏培养皿中 中。在进行之前从所有孵育的卵中取出胚胎 胚胎解剖
    6. 放置含有胚胎的培养皿 在解剖显微镜下,去除周围的膜 胚胎使用镊子和解剖微剪刀(图1a-b)和 使用移液管将其转移到冰上新的冷L-15培养基中
    7. 部分阶段的胚胎和保持期望的发育阶段的胚胎(Hamburger和Hamilton,1992)。

  2. "原始"胚胎解剖
    注意:从这一点开始,胚胎组织的所有解剖步骤将在解剖显微镜下在室温下进行。
    1. 将胚胎在含有冰冷的L-15培养基的培养皿中。有了 直钨丝保持胚胎固定和与 钩形钨针去除尽可能多的间质 而不损害神经管(参见图1b-d)。
    2. 切割 感兴趣的神经管的区域(例如图1e) 钩形针并将神经管件转移到四孔 使用玻璃巴斯德吸管在含有L15培养基的冰上平板。
    3. 重复步骤B1-2到剩余的胚胎,一个时间。如果合适, ?可以合并到四孔板上的相同孔中。

  3. "精细"胚胎剥离
    1. 使用微量吸管拔出硼硅酸盐玻璃毛细管并切割 针的技巧用镊子。通常,尖端大约 直径为0.5mm允许容易地操作解剖组织 (外植体)。
    2. 使用四孔板制备Dispase I溶液 孔,10%FBS/L-15溶液孔和两个L-15培养基孔(洗涤 孔)。使用足以覆盖解剖的溶液体积 组织(每孔500μl溶液通常是足够的)。放在 室温(RT)。将转移来自步骤B的解剖胚胎 ?玻璃巴斯德吸管顺序通过四个孔与一个 在RT下5分钟/孔的孵育期。保持组织在最后 洗涤井,并将其放在冰上。不要孵化组织 Dispase I溶液超过5分钟。
    3. 外套两个10厘米petri 用?2ml FBS的培养皿并在室温下孵育2分钟。删除FBS和 加入冰冷的L-15培养基。将菜肴放在冰上。
    4. 转移一个组织 ?并取出剩余的间充质 组织使用钨针附着到神经管。用一个 直形针以保持组织和钩形针 去除剩余的间充质。一旦所有间质被删除 组织应该看起来"有光泽"和光滑(见图1f)。使用 钩形钨针以切割神经管的期望区域 ?(例如腹侧中线,图1g,中间或背侧) ?神经管的区域)。
    5. 转移解剖组织 (外植体)与玻璃毛细管(步骤C1中制备)在口中混合 吸气管到涂有FBS并含有L-15的四孔板 培养基(约600μl)。改变一个新的涂层培养皿后 第二次组织解剖,以确保培养皿中的培养基 总是冷。

  4. 组织孵育
    1. 根据配方准备床上用品。
    2. 加入16μl 滴到四孔板的孔的底部。孵化在 加湿组织培养箱中37℃孵育25-30min,直至 气泡变得稍微"乳白色"(图1h,左上井)
    3. 加入2滴?50微升垫层溶液到10厘米培养皿的盖子 碟。通过用玻璃毛细管将外植体转移来洗涤外植体 ?嘴抽吸管从一滴床上用品到另一个。
    4. 加 ??20μl的床上用品溶液在干燥的床上用品从步骤上滴下 D2,并将来自步骤D3的洗涤的外植体转移至新的一滴 寝具。外植体将沉到干滴的顶部。位置 外植体使用钨针(见图1i)
    5. 孵化 ?30-60分钟,在37℃的湿润组织培养箱中培养 床上用品下降硬化。加入650μl/孔的生长培养基并在37℃孵育 37℃。可以在任何时间向生长介质中加入可溶性因子 根据实验设置。培养外植体的所需 根据实验设置的时间段。

代表数据

视频1.从鸡卵中分离雏鸡胚胎
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图1.小鸡神经管外植体技术的不同步骤的代表性图像。 a。从鸡蛋隔绝的小鸡胚胎; b。从周围膜分离的鸡胚; C。鸡胚"原始"解剖步骤后; d。钨针保持"原始"解剖胚胎; e。 c中所示的胚胎神经管的解剖的后脑区域; c中的装箱区域对应于e中所示的区域; F。神经管精细解剖后的神经管显示在e。注意,围绕e中的神经管(NT)的间充质(M)现在被完全去除; G。孤立腹侧区神经管,大致对应于f中的方框区; H。在左上孔含有胶原泡的4孔板;一世。包含2个分离的神经管外植体的胶原泡。


食谱

  1. Dispase I溶液
    通过将分散酶I溶解在水中至最终浓度为5mg/ml制备储备溶液 分装溶液,并在-20°C储存一年。
    通过将储备溶液在L-15培养基中稀释至1mg/ml的终浓度来制备Dispase I的工作溶液。
  2. 寝具解决方案
    注意:此解决方案应该是新鲜准备的。
    将8.64μl2.5M NaOH加入到200μlMEM 10x中并涡旋
    解决方案将变为粉红色
    加入1.8ml胶原(3.1mg/ml)并混合
    解决方案将变为杏色
    加入88μlBIC并混合
    最终溶液应为浅粉红色
    将溶液储存在冰上
  3. 生长媒介
    将250ml的DMEM/F12 + Glutamax培养基与250ml的Neurobasal培养基,2.5ml的Glutamax,2.5ml的N2补充物,5ml的B27-补充剂,1.3ml的10mg/ml的BSA储备液, 5ml PEST和4μl2-巯基乙醇。储存在4°C高达一个月

致谢

这项工作的资金由Cancerfonden和Karolinska Institutet提供。该方案改编自Vallstedt等人(2005)和Dias等人(2014)中公开的程序。

参考文献

  1. Dias,J.M.,Alekseenko,Z.,Applequist,J.M.and Ericson,J.(2014)。 Tgfbeta信号调节中枢神经系统中神经干细胞的时间神经发生和效能。 Neuron 84(5):927-939。
  2. Hamburger,V。和Hamilton,H.L。(1992)。 鸡胚发育的一系列正常阶段。 1951. Dev Dyn 195(4):231-272
  3. Lek,M.,Dias,JM,Marklund,U.,Uhde,CW,Kurdija,S.,Lei,Q.,Sussel,L.,Rubenstein,JL,Matise,MP,Arnold,HH,Jessell, ,J.(2010)。 同源异型域反馈电路在腹侧神经图案化过程中支持Shh morphogen梯度的阶梯函数解释。 a> 137(23):4051-4060。
  4. Vallstedt,A.,Klos,J.M。和Ericson,J。(2005)。 脊髓和后脑中少突胶质细胞生成的多个背根源。神经元 45(1):55-67
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引用:Alekseenko, Z., Andersson, E. and Dias, J. M. (2015). Chick Neural Tube Explant Culture. Bio-protocol 5(19): e1608. DOI: 10.21769/BioProtoc.1608.
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