JoVE Journal > Developmental Biology
Summary
Abstract
Introduction
Protocol
Ergebnisse
Discussion
Offenlegungen
Acknowledgements
Materials
Referenzen
Automatische Übersetzung
Please note that all translations are automatically generated. Click here for the English version.
Entwicklungsbiologie

原代小鼠胚胎腭间充质细胞的分离和延时成像，以分析集体运动属性

Published: February 13, 2021
doi:
10.3791/62151
, , ,
1Department of Anatomy and Cell Biology,University of Kansas Medical Center, 2Department of Biological Physics,Eotvos University

Summary

我们提出了一种分离和培养原代小鼠胚胎腭间充质细胞的方案，用于二维（2D）生长和伤口修复测定的延时成像。我们还提供了分析延时成像数据的方法，以确定细胞流形成和方向运动。

Abstract

腭的发育是一个动态过程，它涉及舌头旁边的双侧腭架的垂直生长，然后是舌头上方的抬高和融合。这个过程中的缺陷导致腭裂，这是一种常见的出生缺陷。最近的研究表明，腭架抬高涉及重塑过程，该过程将大陆架的方向从垂直转变为水平。腭架间充质细胞在这种动态重塑中的作用一直难以研究。基于延时成像的定量分析最近已被用于表明原代小鼠胚胎腭间充质（MEPM）细胞可以自我组织成集体运动。定量分析可以识别来自具有上颚抬高缺陷的小鼠模型的突变MEPM细胞的差异。本文描述了从E13.5胚胎中分离和培养MEPM细胞的方法 – 特别是用于延时成像 – 并确定集体运动的各种细胞属性，包括河流形成，形状排列和方向持久性的测量。它假设MEPM细胞可以作为研究腭架间充质在动态抬高过程中的作用的代理模型。这些定量方法将使颅面领域的研究人员能够评估和比较对照细胞和突变细胞的集体运动属性，这将增加对腭架抬高期间间充质重塑的理解。此外，MEPM细胞为研究集体细胞运动提供了一种罕见的间充质细胞模型。

Introduction

腭发育已被广泛研究，因为腭发育缺陷导致腭裂 – 一种常见的出生缺陷，发生在孤立的病例或数百种综合征的一部分1，2。胚胎腭的发育是一个动态过程，涉及胚胎组织的运动和融合。这个过程可以分为四个主要步骤：1）诱导腭架，2）舌头旁边的腭架垂直生长，3）舌头上方腭架的升高，以及4）中线1，3，4的腭架融合。在过去的几十年里，已经鉴定出许多小鼠突变体表现出腭裂5，6，7，8。这些模型的表征表明腭架诱导，增殖和融合步骤存在缺陷;然而，腭架抬高缺陷很少见。因此，了解腭架抬高的动态是一个有趣的研究领域。

对一些具有腭架抬高缺陷的小鼠突变体的仔细分析导致目前的模型显示，腭架的前部区域似乎向上翻转，而腭架的垂直到水平运动或”重塑”发生在上颚的中后区域1，3，4， 9，10，11。腭架的内侧边缘上皮可能启动这种重塑所需的信号传导，然后由腭架间充质驱动。最近，许多研究人员在小鼠模型中发现了腭架抬高延迟，该模型显示涉及腭架的短暂性口腔粘连12，13。间充质重塑涉及细胞的重组，以在水平方向上产生凸起，同时在垂直方向9，10，14上缩回腭架。在提出影响腭架抬高和潜在间充质重塑的几种机制中，有细胞增殖15、16、17、趋化梯度18，和细胞外基质组分19、20。出现了一个重要的问题：在Specc1l-缺陷小鼠中观察到的腭架抬高延迟是否也部分是由于腭架重塑的缺陷，并且这种重塑缺陷是否表现为原代MEPM细胞21行为的内在缺陷？

原代MEPM细胞已被用于颅面领域，涉及基因表达22，23，24，25，26，27，28，29，以及少数涉及增殖30，31和迁移25，31，32的研究，但没有用于集体细胞行为分析。在2D培养和伤口修复测定中对MEPM细胞进行延时成像，以表明MEPM细胞表现出方向运动并形成密度依赖性细胞流 – 集体运动的属性21。此外，Specc1l突变细胞形成更窄的细胞流，并显示出高度可变的细胞迁移轨迹。这种缺乏协调的运动被认为是导致Specc1l突变胚胎13，21的上颚抬高延迟的原因。因此，这些使用原代MEPM细胞的相对简单的测定可以作为研究腭架抬高期间间充质重塑的代理。本文描述了原代MEPM细胞的分离和培养，以及用于2D和伤口修复测定的延时成像和分析。

Protocol

所有涉及动物的实验均按照KUMC机构动物护理和使用委员会批准的协议进行，符合其指南和法规（协议编号：2018-2447）。 1. 收获E13.5胚胎 使用CO2 吸入室或机构动物护理和使用委员会批准的程序对怀孕的雌性小鼠实施安乐死。立即进行解剖。 通过去除皮肤和腹膜来暴露腹腔的下半部分。切除含有E13.5胚胎的子宫的两个角。 将子宫短暂置于预热的37?…

Representative Results

腭架的解剖如图1所示。切口的顺序旨在最大限度地减少组织的滑移。在取下头部（图1A，B）后，下颚被移除（图1B，C）。头部上部（图1C，D）的切口是用来稳定倒置时的组织（图1E）以可视化（图1E，虚线），捏…

Discussion

腭架高程构成垂直到水平的重塑事件1、3、4、9、11。据推测，这种重塑过程需要腭架间充质细胞协调行为。对野生型MEPM细胞的分析表明，这种细胞行为是内在的，可以定量21。因此，这些测定可用于揭示新的和现有的腭裂小鼠模型中的原发性腭架抬高缺陷?…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

该项目部分得到了美国国立卫生研究院拨款DE026172（I.S.）和GM102801（A.C.）的支持。I.S.还得到了生物医学卓越研究中心（COBRE）赠款（国家普通医学科学研究所P20 GM104936），堪萨斯州IDeA生物医学研究卓越网络赠款（国家普通医学科学研究所P20 GM103418）和堪萨斯州智力和发育障碍研究中心（KIDDRC）赠款（U54 Eunice Kennedy Shriver国家儿童健康与人类发展研究所）的部分支持， HD090216）。

Materials

Beaker, 250 mL (x2) Fisher Scientific FB-100-250
CO2 Matheson Gas UN1013
Conical tubes, 15 mL (x1) Midwest Scientific C15B
Debian operating system computational analysis of time-lapse images
Dulbecco's Modified Eagles Medium/High Glucose with 4 mM L-Glutamine and Sodium Pyruvate Cytiva Life Sciences SH30243.01
EtOH, 100% Decon Laboratories 2701
EVOS FL Auto ThermoFisher Scientific AMAFD1000
EVOS Onstage Incubator ThermoFisher Scientific AMC1000
EVOS Onstage Vessel Holder, Multi-Well Plates ThermoFisher Scientific AMEPVH028
Fetal Bovine Serum Corning 35-010-CV
Fine point #5 Stainless Steel Forceps (x2) Fine Science Tools 11295-10 Dissection
Instrument sterilizer bead bath Fine Science Tools 18000-45
Microcetrifuge tubes, 1.5mL Avant 2925
Micro-Dissecting Stainless Steel Scissors, Straight Roboz RS-5910 Dissection
NucBlue (Hoechst) Live Ready Probes ThermoFisher Scientific R37605
Penicillin Streptomycin Solution, 100x Corning 30-002-CI
Silicone Insert, 2-well Ibidi 80209
Small Perforated Stainless Steel Spoon Fine Science Tools MC17C Dissection
Spring Scissors, 4 mm Fine Science Tools 15018-10
Sterile 10 cm dishe(s) Corning 430293
Sterile 12-well plate(s) PR1MA 667512
Sterile 6-well plate(s) Thermo Fisher Scientific 140675
Sterile PBS Corning 21-031-CV
Sterile plastic bulb transfer pipette ThermoFisher Scientific 202-1S
Trypsin, 0.25% ThermoFisher Scientific 25200056
DOWNLOAD MATERIALS LIST

Referenzen

  1. Bush, J. O., Jiang, R. Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development. Development. 139 (2), 231-243 (2012).
  2. Mossey, P. A., Little, J., Munger, R. G., Dixon, M. J., Shaw, W. C. Cleft lip and palate. Lancet. 374 (9703), 1773-1785 (2009).
  3. Lan, Y., Xu, J., Jiang, R. Cellular and molecular mechanisms of palatogenesis. Current Topics in Developmental Biology. 115, 59-84 (2015).
  4. Li, C., Lan, Y., Jiang, R. Molecular and cellular mechanisms of palate development. Journal of Dental Research. 96 (11), 1184-1191 (2017).
  5. Gritli-Linde, A. The etiopathogenesis of cleft lip and cleft palate: usefulness and caveats of mouse models. Current Topics in Developmental Biology. 84, 37 (2008).
  6. Meng, L., Bian, Z., Torensma, R., Vonden Hoff, J. W. Biological mechanisms in palatogenesis and cleft palate. Journal of Dental Research. 88 (1), 22-33 (2009).
  7. Dixon, M. J., Marazita, M. L., Beaty, T. H., Murray, J. C. Cleft lip and palate: understanding genetic and environmental influences. Nature Reviews Genetics. 12 (3), 167-178 (2011).
  8. Kousa, Y. A., Schutte, B. C. Toward an orofacial gene regulatory network. Developmental Dynamics. 245 (3), 220-232 (2016).
  9. Jin, J. Z., et al. Mesenchymal cell remodeling during mouse secondary palate reorientation. Developmental Dynamics. 239 (7), 2110-2117 (2010).
  10. Yu, K., Ornitz, D. M. Histomorphological study of palatal shelf elevation during murine secondary palate formation. Developmental Dynamics. 240 (7), 1737-1744 (2011).
  11. Chiquet, M., Blumer, S., Angelini, M., Mitsiadis, T. A., Katsaros, C. Mesenchymal remodeling during palatal shelf elevation revealed by extracellular matrix and F-actin expression patterns. Frontiers in Physiology. 7, 392 (2016).
  12. Paul, B. J., et al. ARHGAP29 mutation is associated with abnormal oral epithelial adhesions. Journal of Dental Research. 96 (11), 1298-1305 (2017).
  13. Hall, E. G., et al. SPECC1L regulates palate development downstream of IRF6. Human Molecular Genetics. 29 (5), 845-858 (2020).
  14. Walker, B. E., Fraser, F. C. Closure of the secondary palate in three strains of mice. Journal of Embryology and Experimental Morphology. 4 (2), 176-189 (1956).
  15. Jin, J. Z., Li, Q., Higashi, Y., Darling, D. S., Ding, J. Analysis of Zfhx1a mutant mice reveals palatal shelf contact-independent medial edge epithelial differentiation during palate fusion. Cell Tissue Research. 333 (1), 29-38 (2008).
  16. Kouskoura, T., et al. The etiology of cleft palate formation in BMP7-deficient mice. PLoS One. 8 (3), 59463 (2013).
  17. Lan, Y., Zhang, N., Liu, H., Xu, J., Jiang, R. Golgb1 regulates protein glycosylation and is crucial for mammalian palate development. Development. 143 (13), 2344-2355 (2016).
  18. He, F., et al. Wnt5a regulates directional cell migration and cell proliferation via Ror2-mediated noncanonical pathway in mammalian palate development. Development. 135 (23), 3871-3879 (2008).
  19. Lan, Y., Qin, C., Jiang, R. Requirement of hyaluronan synthase-2 in craniofacial and palate development. Journal of Dental Research. 98 (12), 1367-1375 (2019).
  20. Yonemitsu, M. A., Lin, T. Y., Yu, K. Hyaluronic acid is required for palatal shelf movement and its interaction with the tongue during palatal shelf elevation. Entwicklungsbiologie. 457 (1), 57-68 (2020).
  21. Goering, J. P., et al. SPECC1L-deficient palate mesenchyme cells show speed and directionality defect. Scientific Reports. 11 (1), 1452 (2021).
  22. Fantauzzo, K. A., Soriano, P. PI3K-mediated PDGFRalpha signaling regulates survival and proliferation in skeletal development through p53-dependent intracellular pathways. Genes and Development. 28 (9), 1005-1017 (2014).
  23. Vasudevan, H. N., Soriano, P. SRF regulates craniofacial development through selective recruitment of MRTF cofactors by PDGF signaling. Developmental Cell. 31 (3), 332-344 (2014).
  24. Vasudevan, H. N., Mazot, P., He, F., Soriano, P. Receptor tyrosine kinases modulate distinct transcriptional programs by differential usage of intracellular pathways. Elife. 4, 07186 (2015).
  25. Gao, L., et al. 2,3,7,8-Tetrachlorodibenzo-p-dioxin and TGFbeta3-mediated mouse embryonic palatal mesenchymal cells. Dose Response. 17 (1), 1559325818786822 (2019).
  26. Iyyanar, P. P. R., Nazarali, A. J. Hoxa2 inhibits bone morphogenetic protein signaling during osteogenic differentiation of the palatal mesenchyme. Frontiers in Physiology. 8, 929 (2017).
  27. Jiang, Z., Pan, L., Chen, X., Chen, Z., Xu, D. Wnt6 influences the viability of mouse embryonic palatal mesenchymal cells via the beta-catenin pathway. Experimental and Therapeutic Medicine. 14 (6), 5339-5344 (2017).
  28. Liu, X., et al. Negative interplay of retinoic acid and TGF-beta signaling mediated by TG-interacting factor to modulate mouse embryonic palate mesenchymal-cell proliferation. Birth Defects Research Part B: Developmental and Reproductive Toxicology. 101 (6), 403-409 (2014).
  29. Bush, J. O., Soriano, P. Ephrin-B1 forward signaling regulates craniofacial morphogenesis by controlling cell proliferation across Eph-ephrin boundaries. Genes & Development. 24 (18), 2068-2080 (2010).
  30. Mo, J., Long, R., Fantauzzo, K. A. Pdgfra and Pdgfrb genetically interact in the murine neural crest cell lineage to regulate migration and proliferation. Frontiers in Physiology. 11, 588901 (2020).
  31. He, F., Soriano, P. A critical role for PDGFRalpha signaling in medial nasal process development. PLoS Genetics. 9 (9), 1003851 (2013).
  32. Fantauzzo, K. A., Soriano, P. Generation of an immortalized mouse embryonic palatal mesenchyme cell line. PLoS One. 12 (6), 0179078 (2017).
  33. Wu, K., Gauthier, D., Levine, M. D. Live cell image segmentation. IEEE Transactions on Biomedical Engineering. 42 (1), 1-12 (1995).
  34. Neufeld, Z., et al. The role of Allee effect in modelling post resection recurrence of glioblastoma. PLoS Computational Biology. 13 (11), 1005818 (2017).
  35. Zamir, E. A., Czirok, A., Rongish, B. J., Little, C. D. A digital image-based method for computational tissue fate mapping during early avian morphogenesis. Annals of Biomedical Engineering. 33 (6), 854-865 (2005).
  36. Czirok, A., et al. Optical-flow based non-invasive analysis of cardiomyocyte contractility. Scientific Reports. 7 (1), 10404 (2017).
  37. Biggs, L. C., et al. Interferon regulatory factor 6 regulates keratinocyte migration. Journal of Cell Science. 127, 2840-2848 (2014).
  38. Czirok, A., Varga, K., Mehes, E., Szabo, A. Collective cell streams in epithelial monolayers depend on cell adhesion. New Journal of Physics. 15, 75006 (2013).
  39. Szabo, A., et al. Collective cell motion in endothelial monolayers. Physical Biology. 7 (4), 046007 (2010).
  40. Gulyas, M., Csiszer, M., Mehes, E., Czirok, A. Software tools for cell culture-related 3D printed structures. PLoS One. 13 (9), 0203203 (2018).
  41. Soderholm, J., Heald, R. Scratch n’ screen for inhibitors of cell migration. Chemistry & Biology. 12 (3), 263-265 (2005).
  42. Riahi, R., Yang, Y., Zhang, D. D., Wong, P. K. Advances in wound-healing assays for probing collective cell migration. Journal of Laboratory Automation. 17 (1), 59-65 (2012).
  43. Svensson, C. M., Medyukhina, A., Belyaev, I., Al-Zaben, N., Figge, M. T. Untangling cell tracks: Quantifying cell migration by time lapse image data analysis. Cytometry Part A. 93 (3), 357-370 (2018).
  44. Fantauzzo, K. A., Soriano, P. PDGFRbeta regulates craniofacial development through homodimers and functional heterodimers with PDGFRalpha. Genes & Development. 30 (21), 2443-2458 (2016).
  45. Rafi, S. K., et al. Anti-epileptic drug topiramate upregulates TGFβ1 and SOX9 expression in primary embryonic palatal mesenchyme cells: Implications for teratogenicity. PLoS ONE. , (2021).

Tags

Keywords: Primary Mouse Embryonic Palatal Mesenchyme CellsTime-lapse ImagingCollective Movement AttributesPalatal Shelf ElevationCraniofacial FieldMesenchymal RemodelingEmbryonic Day 13.5Palatal ShellMEPM Culture MediumFine ForcepsStainless Steel ScissorsCell Isolation
check_url/de/62151?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Diesen Artikel zitieren
Goering, J. P., Isai, D. G., Czirok, A., Saadi, I. Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes. J. Vis. Exp. (168), e62151, doi:10.3791/62151 (2021).
Zitat herunterladen
Nachdrucke und Berechtigungen

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image