评估外胚层上皮细胞在颅面发育的信号属性
Summary
本文介绍了组织移植技术的目的是测试头的表面外胚层在颅面部发育中的信令和图案属性。
Abstract
实验胚胎学家了解细胞的命运，在发展和组织的相互作用，调节脊椎动物的图案和形态发生（ 例如，1，2，3，4）作用，帮助禽流胚胎无障碍。在这里，我们说明了一种方法，利用此辅助测试期间面部发展的信令和外胚层组织的图案属性。在这些实验中，我们创建了鹌鹑鸡5或鼠标小鸡6嵌合体移植头的表面外胚层，涉及到同一地区或鸡胚异位地区从鹌鹑或鼠标的上颚。作为供体组织移植到雏鸡的鹌鹑是一个目前在鹌鹑，但不是小鸡细胞的核仁标记的优势，从而使调查来区分主机和捐助组织7。同样，一个重复的元素是在小鼠基因组中是无所不在表示，这使我们能够区分主机和捐助组织在鼠标小鸡嵌合体8。使用鼠标作为供体组织的外胚层，将大大扩展我们对这些组织间的相互作用的理解，因为这将使我们能够测试外胚层的信号来自各种突变胚胎的属性。
Protocol
1。准备供体组织准备文化传媒，增强玻璃引脚，增强钨针。 从壳收集胚胎，在冰冷的PBS洗。 使用一个10毫升的注射器和18号针头从蛋壳指出删除1.0毫升的白蛋白。 制作的外壳用剪刀点上方的一个小孔，然后切一个圆形开口揭露胚胎删除头和地点到DMEM培养液（无血清，室温） 额鼻过程剖析从胚胎（或其他捐助者的网站，如上颌或下颌过?…
Discussion
使用这种移植方法，使我们能够确定外胚层包含信令信息，调节背腹极性和proximodistal扩展上颌。鹌鹑或鼠标外胚层，并在这在许多物种6,11组织中的分子信号的保护成果时使用的相似性，表明这是一个高度保守的信号脊椎动物之间的中心。此外，其他研究者使用了类似的技术来测试头外胚层表面的不同区域的信号特性，并发现存在多种信号， 有助于面对12的形态发生在外胚层中心?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由R01 – DE018234和R01 – DE019638。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| 1x PBS | TEK | TEKZR114 | ||
| DMEM | UCSF | CCFDA003 | ||
| BSA | SIGMA | A7906 | ||
| Dispase | GIBCO | 17105-041 | ||
| 35×10 mm Petri dish | Falcon | 1008 | ||
| No. 5 Dumont forceps | Fine Science Tools | 11252-20 | ||
| Scissors | Fine Science Tools | 14058-11 | ||
| Spring Scissors | Fine Science Tools | 15010-11 | ||
| Needle holder | Fine Science Tools | 26016-12 | ||
| Tungsten Needle | Fine Science Tools | 26000 | ||
| Microcapillary tube | Drummond Scientific Company | 3-000-225-G | ||
| Pasteur Pipets | Fisher | 13-678-6B | ||
| Spring scissors | Fine Science Tools | 15010-11 | ||
| Blade holder | Fine Science Tools | 10052-11 | ||
| Razor blade | Fine Science Tools | 10050-00 |
References
- Noden, D. M. The Role of the Neural Crest in Patterning of Avian Cranial Skeletal, Connective, and Muscle Tissues. Developmental Biology. 96, 144-144 (1983).
- Bronner-Fraser, M., Stern, C. Effects of Mesodermal Tissues on Avian Neural Crest Cell Migration. Developmental Biology. 143, 213-213 (1991).
- Schneider, R. A. Neural crest can form cartilages normally derived from mesoderm during development of the avian head skeleton. Developmental Biology. 208, 441-441 (1999).
- Couly, G. Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head. Development. 129, 1061-1061 (2002).
- Evans, D. J., Noden, D. M. Spatial relations between avian craniofacial neural crest and paraxial mesoderm cells. Dev Dyn. , (2006).
- Hu, D., Marcucio, R., Helms, J. A. A zone of frontonasal ectoderm regulates patterning and growth in the face. Development. 130, 1749-1749 (2003).
- Hu, D., Marcucio, R. S. Unique organization of the frontonasal ectodermal zone in birds and mammals. Dev Biol. 325, 200-200 (2009).
- Le Lièvre, C. S., Le Douarin, N. M. Mesenchymal derivatives of the neural crest: analysis of chimaeric quail and chick embryos. Journal of Embryology and Experimental Morphology. 34, 125-125 (1975).
- Bollag, R. J. Use of a repetitive mouse B2 element to identify transplanted mouse cells in mouse-chick chimeras. Exp Cell Res. 248, 75-75 (1999).
- Korn, M. J., Cramer, K. S. Windowing chicken eggs for developmental studies. J Vis Exp. , (2007).
- Eames, B. F., Schneider, R. A. Quail-duck chimeras reveal spatiotemporal plasticity in molecular and histogenic programs of cranial feather development. Development. 132, 1499-1499 (2005).
- Odent, S. Expression of the Sonic hedgehog (SHH ) gene during early human development and phenotypic expression of new mutations causing holoprosencephaly. Hum Mol Genet. 8, 1683-1683 (1999).
- Szabo-Rogers, H. L. Novel skeletogenic patterning roles for the olfactory pit. Development. 136, 219-219 (2009).
Cite This Article
Hu, D., Marcucio, R. S. Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development. J. Vis. Exp. (49), e2557, doi:10.3791/2557 (2011).