the acquisition of chemotactic capabilities, since the Hif pathway
co-regulates Twist, an E-cadherin repressor, and the expression of
CXCR4, the main receptor for CXCL12/Stromal cell-derived factor 1 [9, 11]. Finally, Xenopus NC cells express a wide range of
matrix remodeling factors including several MMPs and ADAMs
[12]. Altogether, this makes the Xenopus NC cells an extremely
relevant model system to study EMT, from its upstream regulators
to its downstream effectors and their impact on cell motility.
From a practical point of view, Xenopus produce a high number
of large embryos, developing externally, allowing complete access
at all times to developmental stages which are hardly accessible in
other models. Furthermore, Xenopus embryos allow for gain and
loss of function studies, by simple microinjection of Morpholino
oligonucleotides, CRISPR/gRNA or mRNA in blastomeres during
segmentation. The use of nontoxic fluorescent dyes permits lineage
tracing during developmental processes. Using this technique, a
cell fate map was generated, allowing reliable targeting of subpopulations of cells, including NC, by injecting specific blastomeres.
Finally, NC are easily extracted from developing embryos to be
grafted into host embryos for in vivo studies, or cultured ex-vivo
in coated Petri dishes in a simple culture medium up to differentiation stages. If dissected at late neurula stage, NC are fully induced
and will spontaneously undergo EMT-driven dispersion and migration during the first few hours following grafts or culture. EMT and
migration of NC cells can be monitored in vivo or ex vivo using
time-lapse cinematography.
In this book chapter, we describe the protocol to extract NC
cells for ex vivo culture and discuss how to monitor and analyze NC
dispersion and migration from time-lapse images.
2 Materials
2.1 Ex-Vivo Culture
of Xenopus Neural
Crest cells
2.1.1 Extracellular Matrix
Preparation
1. Petri dishes (any dish from 35 to 90 mm made of untreated
plastic) or multi-well dishes (e.g., LabTek, Ibidi μSlides 80.821
or equivalent).
2. Purified Fibronectin in solution (Sigma F1141 or equivalent
reference from other providers).
3. Phosphate Buffer Saline (PBS) 1Â: Start with 800 mL of
distilled water add 8 g of NaCl, 0.2 g of KCl, 1.44 g of
Na 2 HPO 4 , 0.24 g of KH 2 PO 4 , adjust the pH to 7.4 with
HCl, add distilled water to a final volume of 1 L.
4. Bovine Serum Albumin (BSA).
5. Heating block.
Using Xenopus Neural Crest to Study EMT
259
co-regulates Twist, an E-cadherin repressor, and the expression of
CXCR4, the main receptor for CXCL12/Stromal cell-derived factor 1 [9, 11]. Finally, Xenopus NC cells express a wide range of
matrix remodeling factors including several MMPs and ADAMs
[12]. Altogether, this makes the Xenopus NC cells an extremely
relevant model system to study EMT, from its upstream regulators
to its downstream effectors and their impact on cell motility.
From a practical point of view, Xenopus produce a high number
of large embryos, developing externally, allowing complete access
at all times to developmental stages which are hardly accessible in
other models. Furthermore, Xenopus embryos allow for gain and
loss of function studies, by simple microinjection of Morpholino
oligonucleotides, CRISPR/gRNA or mRNA in blastomeres during
segmentation. The use of nontoxic fluorescent dyes permits lineage
tracing during developmental processes. Using this technique, a
cell fate map was generated, allowing reliable targeting of subpopulations of cells, including NC, by injecting specific blastomeres.
Finally, NC are easily extracted from developing embryos to be
grafted into host embryos for in vivo studies, or cultured ex-vivo
in coated Petri dishes in a simple culture medium up to differentiation stages. If dissected at late neurula stage, NC are fully induced
and will spontaneously undergo EMT-driven dispersion and migration during the first few hours following grafts or culture. EMT and
migration of NC cells can be monitored in vivo or ex vivo using
time-lapse cinematography.
In this book chapter, we describe the protocol to extract NC
cells for ex vivo culture and discuss how to monitor and analyze NC
dispersion and migration from time-lapse images.
2 Materials
2.1 Ex-Vivo Culture
of Xenopus Neural
Crest cells
2.1.1 Extracellular Matrix
Preparation
1. Petri dishes (any dish from 35 to 90 mm made of untreated
plastic) or multi-well dishes (e.g., LabTek, Ibidi μSlides 80.821
or equivalent).
2. Purified Fibronectin in solution (Sigma F1141 or equivalent
reference from other providers).
3. Phosphate Buffer Saline (PBS) 1Â: Start with 800 mL of
distilled water add 8 g of NaCl, 0.2 g of KCl, 1.44 g of
Na 2 HPO 4 , 0.24 g of KH 2 PO 4 , adjust the pH to 7.4 with
HCl, add distilled water to a final volume of 1 L.
4. Bovine Serum Albumin (BSA).
5. Heating block.
Using Xenopus Neural Crest to Study EMT
259