Download In Ovo Electroporation for Targeting the Somitic Mesoderm

Chapter 5
In Ovo Electroporation for Targeting
the Somitic Mesoderm
Emi Ohata and Yoshiko Takahashi
1
Introduction
The somite is a transient structure present in early vertebrate embryos, giving rise
to a variety of essential tissues including skeletal muscles, dermis, axial bones and
blood vessels. The term “somite” refers to a tissue of spherical structure that forms
by pinching off from the continuous tissue called presomitic mesoderm (PSM,
also called segmental plate in avian embryos). The PSM is recognized as a pair of
longitudinal stripes along the midline of the body. Thus, each somite forms at the
anterior end of PSM, and this process recurs periodically in time and space, generating the segmented pattern of the body along the antero-posterior axis.
Soon after the invention of the in ovo electroporation technique that was
originally applied to the neural tube of chicken embryos (Funahashi et al., 1999;
Momose et al., 1999; Nakamura et al., 2004), somites were also challenged for
electroporation-mediated transgenesis. However, as long as the PSM was targeted,
transgenesis was not successful for unknown reasons. Several years ago, we
achieved somitic transgenesis by targeting PSM precursors (presumptive somitic
cells) of earlier embryos, the cells residing in the epiblast of the anterior primitive
streak (Nakaya et al., 2004; Sato et al., 2002). When development proceeds, these
cells ingress and migrate anteriorly beneath the ectoderm to form the PSM on either
side of the neural tube (Fig. 5.1). We will describe the methods of electroporationmediated transgenesis of early somite/PSM by taking advantage of the dynamic
morphogenetic movement of the presumptive somitic cells.
E. Ohata and Y. Takahashi()
Graduate School of Biological Sciences, Nara Institute of Science and Technology,
Ikoma, Nara 630-0192, Japan
e-mail: [email protected]
H. Nakamura (ed.), Electroporation and Sonoporation in Developmental Biology,
DOI: 10.1007/978-4-431-09427-2_5, © Springer 2009
37
Fig. 5.1 (a) A chicken embryo of stage 8 is visualized with black ink injected in between the
embryo and yolk. (b) Diagrams demonstrate DNA electroporation into developing somites by
taking advantage of mesodermal ingression from the epiblast. Electroporation is carried out with
a stage 8 embryo wherein presumptive somitic cells reside in the epiblast. As development
proceeds, the electroporated cells ingress as a mesoderm inward the embryo, and migrate anteriorly to comprise the PSM by E2. (c) An E3 embryo displays successful transgenesis with
electroporated EGFP cDNA (See Color Plates)
5 In Ovo Electroporation for Targeting the Somitic Mesoderm
2
2.1
39
Procedure
Embryos
Cell fate studies showed that in chicken embryos of stage 8 (Hamburger and
Hamilton, 1951), the presumptive PSM/somite cells are located in the anterior-most
region of the primitive streak just posteriorly to Hensen’s node (Catala et al., 1996;
Psychoyos and Stern, 1996). We here describe the method using stage 8 embryos
as an example. In our experiences, embryos younger than stage 6 are very sensitive
to electric pulses and prone to be easily damaged.
2.2 Devices and Settings for Electroporation
A positive electrode is prepared with a piece of platinum wire with a ‘Z’ shape (diameter: 0.3–0.5 mm). The electrode is held by a micromanipulator (NARISHIGE), and
is connected to the red lead (anode) (Fig. 5.2a). A negative electrode is made from
tungsten, sharpened either by being immersed in hot melting sodium nitrite or by electrolysis with a solution of sodium hydroxide (2M) so that a diameter of the tip becomes
around 40 μm. The prepared electrode is encased in a plastic holder for insulation, and
connected to the black lead (cathode) (Fig. 5.2b). To generate electric pulses, we use
ELECTROPORATOR CUY21. The condition for applying pulses is 4–5 V, 25 ms,
five pulses with 975 ms intervals. These parameters can be optimized for each
experimental situation.
2.3
DNA Preparation
The preparation of DNA plasmids and DNA solution is essentially the same as that
used for other tissues. A DNA cocktail needs to be prepared freshly. Prior to the
electroporation we add Fast Green FCF (Wako) dye with the final concentration of
2% to visualize the DNA solution in embryos.
2.4 Procedures for In Ovo Electroporation
Eggs are incubated horizontally at 38.5°C. When embryos develop to stage 8,
2–3 ml of thin albumen is removed by a syringe needle from the pointed end of egg,
and a window is made on the top of the shell. The embryo is visualized by injecting
black ink into the yolk underneath an embryo. HANKS’ saline is useful to keep the
embryo and electrodes wet.
A piece of vitelline membrane is removed by a tungsten needle to expose the anterior
primitive streak (Fig. 5.2e). Subsequently, the platinum electrode (anode) is inserted
Fig. 5.2 A procedure for in ovo electroporation to target presumptive PSM/somite in chicken
embryos of stage 8. (a) A setting for in ovo electroporation. A platinum electrode is held with a
manipulator and connected to the anode. (b) A sharpened tungsten electrode is encased in a plastic
holder, and connected to the cathode. (c, d) A platinum electrode is inserted into the yolk, and
adjust the manipulator to locate the tip of the electrode underneath the anterior primitive streak.
(e) After a DNA solution is laid on top of the area to be targeted, a tungsten electrode is positioned
nearby, followed by application of electric pulses. (f) Relative positions between the DNA
solution, embryo, and two electrodes. * Hensen’s node (See Color Plates)
5
In Ovo Electroporation for Targeting the Somitic Mesoderm
41
into the yolk (Fig. 5.2c) with the tip of the electrode positioned underneath the anterior
primitive streak (Fig. 5.2d). Using a glass capillary, a DNA solution is laid on the epiblast
of the anterior primitive streak. Immediately, the tungsten electrode (cathode) is carefully
placed on the area to be targeted, and electric pulses are applied (Fig. 5.2f). Bubbles
come out near the tip of the tungsten electrode, and it is a sign of the electric current
generated. Importantly, both electrodes should be positioned closely to but not in a direct
contact with the target since a direct contact would cause severe burn or malformation
of tissues. It is also important to correctly locate the area of presumptive somite in the
primitive streak because targeting more anterior or posterior regions would result in
transgenesis of the neural tube or lateral plate, respectively. The anterior primitive streak
used for somitic transgenesis also contains presumptive neural tube cells (Catala et al.,
1996; Psychoyos and Stern, 1996). Therefore a successful transgenesis in the somites is
often accompanied with some signals electroporated in the forming neural tube.
Following the electroporation, the platinum electrode is pulled out, and the window on the shell is sealed with plastic tape. Manipulated embryos are re-incubated
until they are sacrificed.
When we use pCAGGS-EGFP, signals of EGFP become detectable ~4 h after
electroporation, and the signals persist for three to four days at most (E4 or E5)
(Fig. 5.1c). The electroporated cells are mosaic in developing somites with 30%–50%
of the total population of cells.
3
Comments
We have described the method for somitic transgenesis that takes advantage of the
ingression of presumptive mesoderm from the epiblast. However, if a product of electroporated gene affects the ingression process, one cannot investigate the role of the
gene at later stages including somitogenesis. To circumvent such problems, we have
optimized the condition of tetracycline-inducible expression of electroporated genes
as described in Sato and Takahashi in this book (see also Watanabe et al., 2007).
For somitic electroporation, there is another method reported by Scaal et al., which
is useful for targeting a specific region within a formed somite, such as dermomyotome. In this case, a DNA solution is injected into a somitocoele of formed somite and
electric pulses are applied with electrodes positioned laterally (Scaal et al., 2004).
4
4.1
Troubleshooting
High Lethality of Electroporated Embryos
• Decrease voltages and/or the number of pulses to a milder condition.
• Check whether tungsten or platinum electrodes are in a direct contact with an
embryo. They should not touch the targeting tissue.
42
E. Ohata and Y. Takahashi
• Sharpen the tip of a tungsten electrode to have a smaller diameter.
• Insert the platinum electrode more deeply in the yolk. This would increase a
resistance between two electrodes.
• Avoid using embryos earlier than stage 6.
4.2
Electroporated Cells Are Seen More
in the Neural Tube than in PSM
• Target a region more posteriorly further from Hensen’s node. The region around
Hensen’s node contains more of neural tube precursors than the region used for
the somitic transgenesis.
• The efficiency of somitic transgenesis decreases if embryos older than stage 9
are used.
References
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in the avian embryo: while regressing, Hensen’s node lays down the notochord and floor plate
thus joining the spinal cord lateral walls. Development 122, 2599–2610.
Funahashi, J., Okafuji, T., Ohuchi, H., Noji, S., Tanaka, H., Nakamura, H. (1999). Role of Pax-5
in the regulation of a mid-hindbrain organizer’s activity. Dev Growth Differ 41, 59–72.
Hamburger, V., Hamilton, H. L. (1951). A series of normal stages in the development of the chick
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Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K., Yasuda, K. (1999). Efficient
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