Rediscovering the chick embryo as a model to study retinal development

Abstract

The embryonic chick occupies a privileged place among animal models used in developmental studies. Its rapid development and accessibility for visualization and experimental manipulation are just some of the characteristics that have made it a vertebrate model of choice for more than two millennia. Until a few years ago, the inability to perform genetic manipulations constituted a major drawback of this system. However, the completion of the chicken genome project and the development of techniques to manipulate gene expression have allowed this classic animal model to enter the molecular age. Such techniques, combined with the embryological manipulations that this system is well known for, provide a unique toolkit to study the genetic basis of neural development. A major advantage of these approaches is that they permit targeted gene misexpression with extremely high spatiotemporal resolution and over a large range of developmental stages, allowing functional analysis at a level, speed and ease that is difficult to achieve in other systems. This article provides a general overview of the chick as a developmental model focusing more specifically on its application to the study of eye development. Special emphasis is given to the state of the art of the techniques that have made gene gain- and loss-of-function studies in this model a reality. In addition, we discuss some methodological considerations derived from our own experience that we believe will be beneficial to researchers working with this system.

Figure 1

Schematic representation of vertebrate eye development. (A) Specification of the eye field within the anterior neural plate. (B) Formation of the optic vesicle. (C) Specification of the RPE, neural retina and optic stalk domains within the optic vesicle and formation of the lens placode from the surface ectoderm. (D) Formation of the optic cup and the lens vesicle. (E) Mature optic cup and lens. (F) Organization of the mature retina. Abbreviations: C: Cornea; ef: eye field; gcl: ganglion cell layer; inl: inner nuclear layer; ipl: inner plexiform layer; L: lens; LP: lens placode; LV: lens vesicle; MS: mesenchyme; NR: neural retina; ON: optic nerve; onl: outer nuclear layer; opl: optic plexiform layer; OS: optic stalk; OV: optic vesicle; RPE: retinal pigment epithelium; S: sclera; SE: surface ectoderm. Modified with permission from Adler and Canto-Soler, 2007.

Figure 2

Recommended guidelines to ensure egg quality for research purposes. SPF eggs should be screened for the absence of ASLV viral particles and endogenous viral protein expression. Once an appropriate source of eggs is selected, screening should be performed routinely to ensure quality maintenance. After experimental treatment, infection can be assessed by immunohistochemistry (IHC) or in situ hybridization (ISH) designed to detect a specific RCAS subgroup, or by the use of reporter constructs. Originally published in McNally et al., 2010. Reproduced with permission.

Figure 3

Gain of function by retroviral gene expression. Viral solution is injected into the anterior neural tube of ED1.5 chicken embryos (A) or in the vitreal cavity of ED3 chicken embryos (B). (C-D) Transversal section of the retina of an ED12 embryo that was injected with RCAS construct expressing GFP on ED3. (C) Shows GFP expression (green) throughout the layers of the retina; (D) detection of viral infection by an antibody against RCAS subgroup A envelop protein (magenta). Abbreviations: gcl: ganglion cell layer; inl: inner nuclear layer; onl: outer nuclear layer.

Figure 4

Loss-of-function by morpholino antisense oligonucleotides. (A-B) Setting and electrodes utilized for electroporation at ED1.5. (C-D) Setting and electrodes utilized for electroporation at ED3-4. (E-G) Images from embryos electroporated with fluorescein-labeled morpholinos at ED1.5. (E) Dorsal view of a whole mounted embryo fixed immediately after electroporation; (F) Side view of a whole-mounted embryo fixed 24 h after electroporation; (G) transversal section of the eye of an embryo fixed 24 h after electroporation. Arrows indicate morpholino incorporation (green) into retinal progenitor cells. (H) Flat mount of a retina electroporated with morpholino at ED4 and collected 24 h later. Abbreviations: cf: choroid fissure; onh: optic nerve head.