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. 2015 Feb 24:9:45.
doi: 10.3389/fnins.2015.00045. eCollection 2015.

Genetic manipulation of reptilian embryos: toward an understanding of cortical development and evolution

Tadashi Nomura  1 , Wataru Yamashita  2 , Hitoshi Gotoh  3 , Katsuhiko Ono  3
Affiliations

Genetic manipulation of reptilian embryos: toward an understanding of cortical development and evolution

Tadashi Nomura et al. Front Neurosci. .

Abstract

The mammalian neocortex is a remarkable structure that is characterized by tangential surface expansion and six-layered lamination. However, how the mammalian neocortex emerged during evolution remains elusive. Because all modern reptiles have a homolog of the neocortex at the dorsal pallium, developmental analyses of the reptilian cortex are valuable to explore the origin of the neocortex. However, reptilian cortical development and the underlying molecular mechanisms remain unclear, mainly due to technical difficulties with sample collection and embryonic manipulation. Here, we introduce a method of embryonic manipulations for the Madagascar ground gecko and Chinese softshell turtle. We established in ovo electroporation and an ex ovo culture system to address neural stem cell dynamics, neuronal differentiation and migration. Applications of these techniques illuminate the developmental mechanisms underlying reptilian corticogenesis, which provides significant insight into the evolutionary steps of different types of cortex and the origin of the mammalian neocortex.

Keywords: amniotes; cortex; evolution; ex vivo culture; in ovo electroporation; reptiles.

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Figures

Figure 1
Figure 1
Unique characteristics of Madagascar ground gecko and Chinese softshell turtle. (A) Phylogenic position of the gecko and turtle among amniotes. Lepidosaurs include sphenodon, snake, lizard and gecko, whereas archosaurs include turtle, crocodile and bird. (B,C) Young individuals of Madagascar ground gecko (Paroedura pictus) (B) and Chinese softshell turtle (Pelodiscus sinensis) (C). (D) Developmental rates of Paroedura pictus and Gallus gallus (chick). Equivalent developmental stages are based on limb bud and cranial morphology (Wise et al., 2009). Representative developmental events include 1: hindlimb bud develops, 2: hindlimb bud becomes larger than forelimb bud, 4: autopodium develops discrete paddle shape, 5: zeugopodium and stylopodium become distinct, 6: digits develop, 9: phalanges develop, 10: claws develop, 11: scale formation and pigmentation, and 12: hatching. Detailed staging criteria are described in Wise et al. (2009).
Figure 2
Figure 2
In ovo electroporation of gecko embryos. (A) Experimental equipment. (B) Needle-type electrodes (CUY200S). (C) Two P. pictus eggs incubated in a small tapper with vermiculite. (D) Sterilization of the egg with 70% ethanol and a cotton stick. (E) HBSS was dropped through the hole of the shell. (F) The window was opened with fine forceps. (G) An illustration showing the position of the electrodes on the embryo. (H) High magnification of an electroporated embryo. Green-colored DNA solution was injected in the left lateral ventricle. (I) The window was sealed with a cover glass. (J) Incubation of operated embryos in the container.
Figure 3
Figure 3
In ovoelectroporation of turtle embryos. (A) Turtle eggs in the delivery packet. Before transferring the eggs, the top of the shell was marked to maintain an upside-down orientation. (B) Tools for surgical manipulation. 1: A depression slide, 2: micro scissors, 3: forceps, 4: mini drill (pinvise), 5: hand-made egg stand, and 6: a metal file. (C) A small scar was made on the shell with a metal file. (D) A pin vise was used to drill the surface of the egg. (E) HBSS was dropped through the small window. (F) The window was sealed with a cover glass after electroporation.
Figure 4
Figure 4
Ex ovo culture system for reptilian embryos. (A) Turtle embryos were opened in HBSS. (B) A turtle embryo in which a DNA solution (green color) was injected into the lateral ventricle. (C–E) Incubation of gecko (C) and turtle (D,E) embryos in the whole embryo culture system. Electroporated embryos were cultured in glass vials filled with HBSS. Embryo containing vials were inserted into the rotator to supply oxygen continuously. To avoid crushing the embryos, the rotating wheel was not used during the culture.
Figure 5
Figure 5
GFP expression in the developing gecko and turtle cortex. (A) Developing gecko embryo after electroporation. The image was captured using an iPhone4S camera through a magnifier. (B) Gecko embryos at 4 days after electroporation. GFP was expressed at the dorsal part of the telencephalon (arrows). (C) GFP expression in the cortical neural stem/progenitor cells. Mitotic GFP-positive cells were labeled with an anti-phospho histoneH3 (PH3) antibody (red arrows). A GFP-positive cell at the outside of the ventricular zone was not mitotic (white arrow). (D) The distribution of GFP-positive cells in the gecko cortex at 7 days after electroporation. Arrows indicate migrating neurons (E,F) GFP expression in the developing turtle cortex at 4 days after electroporation. Arrows indicate migrating neurons. (G–I) GFP expression in the gecko cortex at 1 month after electroporation. VZ, ventricular zone; NL, neuronal layer; OB, olfactory bulb; DC, dorsal cortex; DVR, dorsal ventricular ridge. Scale bars: 25 μm (C,F), 50 μm (E).
Figure 6
Figure 6
The expression of Tbr2 in the developing gecko cortex. (A) Western blot with anti-mouse Tbr2 antibody. The left panel shows lysate from HEK293 cells transfected with the expression vector for mouse Tbr2. The control lane was whole cell lysate without transfection. A major band was detected at the predicted molecular weight (72 kD) for mouse Tbr2. A slightly lager band was possibly due to post-translational modification. The right panel shows western blot of embryonic turtle (st17), gecko (d.p.o.18), chick (E7), and mouse (E14) brain lysate. Together with the bands of predicted molecular weight (72 kD), additional larger bands were detected in all examined species. (B–D) Immunohistochemistry of the developing gecko cortex (d.p.o. 18) with anti-Tbr2 antibody. Tbr2-positive cells were detected at the basal side of the ventricular zone (white arrows). (C) Tbr2-positive cells did not overlap with BrdU-incorporated cells. (D) All BrdU-incorporated cells were Sox2-positive. Detailed immunohistochemistry and BrdU incorporation protocols were described previously (Nomura et al., 2013a).
Figure 7
Figure 7
Characteristics of migrating neurons in the developing amniote pallia. (A–D) Electroporation of GFP-expression vector into the developing mouse (A), gecko (B), turtle (C), and chick (D) pallia. (E–L) Distribution and morphology of GFP-positive migrating neurons in the mouse neocortex (E,I) and the gecko (F,J), turtle (G,K) and chick (H,L) pallia. (M–P) Contour graphs of the longest process orientation of mouse (M; the data were taken from the cortical plate), gecko (N), turtle (O), and chick (P). The angles of the processes were calculated against the ventricular plane. Each contour line represents the number of cells. (Q–T) The expression of CTIP2 in RFP-positive pallial neurons in the developing gecko cortex (white arrows in R–T).
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