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Biology

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Published: April 12th, 2013

DOI:

10.3791/50248

1Department of Craniofacial Biology, University of Colorado Denver Anschutz Medical Campus, 2Department of Cell and Developmental Biology, University of Colorado Denver Anschutz Medical Campus

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.

Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

1. Prepare Dispase II

  1. Prepare fresh Hepes-buffered Saline (HBS) (50 mM Hepes/KOH pH 7.4, 150 mM NaCl). Add 2 ml 0.5 M Hepes/KOH pH 7.4 and 1.2 ml 2.5 M NaCl into 16.8 ml Ultrapure H2O.
  2. Make 10 mg/ml Dispase II. Weigh 0.2 g Dispase II (Roche, Cat# 4942078001) and dissolve in 20 ml HBS. Prepare 1.0 ml aliquots in individual Eppendorf tubes and store at -20 °C for long-term storage.
  3. Dilute 10 mg/ml Dispase II 1:10 in PBS before use. Put the diluted Dispase II on ice.
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After Dispase II treatment, the ectoderm of the embryo tends to be loose. The facial prominences should be intact after dissection (Figure 2A). The isolated facial ectoderm is clear and free of mesenchyme tissue (Figure 2B). To determine the efficacy of the protocol and to detect cross contamination we assayed for forebrain expressed Zic39, ectodermal specific cdh15 and mesenchymal specific sox1010 using reverse transcriptase PCR. Our studies confirmed th.......

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This protocol provides a straightforward method to separate embryonic mouse facial ectoderm and mesenchyme based on an initial Dispase II treatment step. In previous studies, we have performed dissection of the facial prominences prior to Dispase II treatment, but we have consistently found that the mesenchyme becomes "sticky" and more difficult to manipulate. Our new protocol avoids this problem. Combination Dispase II treatment with gentle physical force by pipetting up and down makes separation of ectoderm and mesench.......

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The authors would like to thank Irene Choi for illustrating embryo heads in Fig 1 and the other members of the laboratory for help and discussion. This work is supported by NIH grant DE012728 (T.W.)

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Name of Reagent/Material Company Catalog Number Comments
Dispase II (neutral protease, grade II) Roche 4942078001 Make 10 mg/ml Dispase II stock solution in HBS. Aliquot and store at -20 °C .
RNAlater Ambion AM7021  

  1. Wilkie, A. O., Morriss-Kay, G. M. Genetics of craniofacial development and malformation. Nat. Rev. Genet. 2, 458-468 (2001).
  2. Gritli-Linde, A. Chapter 2 - The Etiopathogenesis of cleft lip and cleft palate: usefulness and caveats of mouse models. Current Topics in Developmental Biology. 84, 37-138 (2008).
  3. Schutte, B. C., Murray, J. C. The many faces and factors of orofacial clefts. Human Molecular Genetics. 8, 1-7 (1999).
  4. Chai, Y., Maxson, R. E. Recent advances in craniofacial morphogenesis. Dev. Dyn. 235, 2353-2375 (2006).
  5. Jiang, R., Bush, J. O., Lidral, A. C. Development of the upper lip: Morphogenetic and molecular mechanisms. Dev. Dyn. 235, 1152-1166 (2006).
  6. Reid, B. S., Yang, H., Melvin, V. S., Taketo, M. M., Williams, T. Ectodermal WNT/β-catenin signaling shapes the mouse face. Developmental Biology. 349, 261-269 (2011).
  7. Feng, W., et al. Spatial and temporal analysis of gene expression during growth and fusion of the mouse facial prominences. PLoS ONE. 4, e8066 (2009).
  8. Chia, R., Achilli, F., Festing, M. F., Fisher, E. M. The origins and uses of mouse outbred stocks. Nat. Genet. 37 (11), 1181-1186 (2005).
  9. Nagai, T., Aruga, J., Takada, S., et al. The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation. Developmental Biology. 182, 299-313 (1997).
  10. Britsch, S., et al. The transcription factor Sox10 is a key regulator of peripheral glial development. Genes Dev. 15 (1), 66-78 (2001).

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