Chick Embryo Eyes Research Articles

Atlas Toolkit: Fast registration of 3D morphological datasets in the absence of landmarks.

Image registration is a gateway technology for Developmental Systems Biology, enabling computational analysis of related datasets within a shared coordinate system. Many registration tools rely on landmarks to ensure that datasets are correctly aligned; yet suitable landmarks are not present in many datasets. Atlas Toolkit is a Fiji/ImageJ plugin collection offering elastic group-wise registration of 3D morphological datasets, guided by segmentation of the interesting morphology. We demonstrate the method by combinatorial mapping of cell signalling events in the developing eyes of chick embryos, and use the integrated datasets to predictively enumerate Gene Regulatory Network states.

Effect of Ultrahigh Frequency Radiation Emitted from 2G Cell Phone on Developing Lens of Chick Embryo: A Histological Study

A Mobile phone in operation emits a pulsed radiofrequency electromagnetic field which is absorbed into the user’s body particularly the head region. Contradictory scientific reports on the health effect of nonionizing radiations on biological tissues have prompted to undertake the present study to evaluate the damage in the developing lens of a chick embryo following exposure to radiation emitted from a 2G cell phone. Fertilized chick embryos were incubated in two groups in a standard egg incubator. The experiment group was exposed to radiation emitted from a 2G cell phone. On completion of scheduled duration, the embryos were collected and processed for routine histological studies. The 9th to 12th day chick embryo eyes were processed for assessment of DNA damage using the alkaline comet assay technique. The lens thickness and the equatorial diameter were measured using oculometer and statistically compared for both groups. In the present study, the exposure of chick embryos to a 2G cell phone caused structural changes in lens epithelial cells, formation of cystic cells and spaces, distortion of lens fibers, and formation of posterior aberrant nuclear layer. The DNA damage in the developing eyes of the experiment group assessed by comet assay was highly significant.

Ectopic expression of transcription factor AP‐2δ in developing retina: effect on PSA‐NCAM and axon routing

Retinal ganglion cells transmit the visual signal from the retina to the brain. We have previously shown that the activator protein 2 (AP-2)δ (TFAP2D) transcription factor is expressed in one third of ganglion cells in developing retina suggesting a specialized role for these AP-2δ-expressing cells. Here, we address the role of AP-2δ in retina by in ovo electroporation of RCAS/AP-2δ retroviral constructs into the eyes of chick embryos at day 2 of gestation. Ectopic expression of AP-2δ does not affect lineage differentiation in the developing retina. However, immunostaining of retinal tissue with markers associated with axonal growth such as growth-associated protein 43 and polysialic acid-neural cell adhesion molecule (PSA-NCAM) demonstrates axonal misrouting and abnormal axonal bundling. Treatment of AP-2δ-misexpressing retinal cell cultures with endoneuraminidase, an enzyme that removes PSA from NCAM, decreases AP-2δ-induced axonal bundling. Our data suggest a role for AP-2δ in polysialylation of NCAM, with ectopic expression of AP-2δ resulting in premature bundling of emerging axons and misrouting of axons. We propose that expression of AP-2δ in a subset of ganglion cells contributes to the fine-tuning of axonal growth in the developing retina.

Chick <em>ex ovo</em> Culture and <em>ex ovo</em> CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci. For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used(1-4), no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay(5,6), (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye(7-9), (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM(10-12), (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice(13) as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM(14). In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications.

3-Dimensional modelling of chick embryo eye development and growth using high resolution magnetic resonance imaging

Magnetic resonance imaging (MRI) is a powerful tool for generating 3-dimensional structural and functional image data. MRI has already proven valuable in creating atlases of mouse and quail development. Here, we have exploited high resolution MRI to determine the parameters necessary to acquire images of the chick embryo eye. Using a 9.4 Tesla (400 MHz) high field ultra-shielded and refrigerated magnet (Bruker), MRI was carried out on paraformaldehyde-fixed chick embryos or heads at E4, E6, E8, and E10. Image data were processed using established and custom packages (MRICro, ImageJ, ParaVision, Bruker and mri3dX). Voxel dimensions ranged from 62.5 μm to 117.2 μm. We subsequently used the images obtained from the MRI data in order to make precise measurements of chick embryo eye surface area, volume and axial length from E4 to E10. MRI was validated for accurate sizing of ocular tissue features by direct comparison with previously published literature. Furthermore, we demonstrate the utility of high resolution MRI for making accurate measurements of morphological changes due to experimental manipulation of chick eye development, thereby facilitating a better understanding of the effects on chick embryo eye development and growth of such manipulations. Chondroitin sulphate or heparin were microinjected into the vitreous cavity of the right eyes of each of 3 embryos at E5. At E10, embryos were fixed and various eye parameters (volume, surface area, axial length and equatorial diameter) were determined using MRI and normalised with respect to the un-injected left eyes. Statistically significant alterations in eye volume (p < 0.05; increases with chondroitin sulphate and decreases with heparin) and changes in vitreous homogeneity were observed in embryos following microinjection of glycosaminoglycans. Furthermore, in the heparin-injected eyes, significant disturbances at the vitreo-retinal boundary were observed as well as retinal folding and detachment confirming histological observations. These data reveal the utility and superiority of MRI for producing images enabling quantification of experimentally induced changes in eye volume and structure. The results indicate that MRI is an important tool that could become a routine approach for rapid and sensitive phenotypic analysis of normal chick ocular development and morphology as well as potentially the effects of surgical or genetic manipulations of chick embryo eyes in live embryos in ovo.

Pharmacological vitreous remodelling through non‐enzymatic disruption of the protein matrix

Abstract Purpose To study the impact of inhibiting lysyl oxidase enzyme on vitreous extracellular matrix (ECM) proteins. Lysyl oxidase (LO) plays a critical role in the formation and repair of the ECM by oxidizing lysine residues and initiating covalent cross‐links that stabilize fibrous proteins throughout the body. The LO activity in the eye could be an important aspect of vitreous embryogenesis and aging. Methods Eyes (E8) of chick embryos were injected with beta‐aminopropionitrile (BAPN: selective inhibitor of LO) or with saline. Eyes were fixed in glutaraldehyde 2% for TEM, in paraformaldehyde 3% for immunohistochemical staining or the eyes were lysed and prepared for immunoprecipitation (coll II) followed by mass spectrometry (MS: Fourier transform,MALDI‐TOF/TOF and Orbitrap). Results Microscopy showed a homogenous collagen network in the vitreous core and dense collagen fibers in the periphery of saline treated eyes. BAPN treated eyes showed a small triangular area of collagen meshwork immediately behind the lens and scarce collagen fragments in the periphery. Immunohistochemistry revealed positive staining for opticin in the vitreous periphery and for collagen IX in the vitreous centre and periphery in saline treated eyes. Immunostaining was negative for opticin and collagen IX in the BAPN treated eyes. MS identified a dose dependant signal shift in the treated samples for opticin, decorin and collagen IX. Conclusion Inhibiting lysyl oxidase enzyme in the embryonic chick vitreous has a disrupting impact on its ECM assembly as shown by histology. The effect of BAPN on the vitreous ultrastructure could be related to the loss of decorin, opticin and collagen IX as suggested by MS and immunohistochemistry.

Regulation of Eye Size by the Retinal Basement Membrane and Vitreous Body

Congenital high myopia is an early-onset enlargement of the eye globes that carries a high risk for retinal detachment. The genetic basis for congenital high myopia has frequently been connected to mutations in genes encoding extracellular matrix proteins of the vitreous body (VB) and the inner limiting membrane (ILM). Why defective or missing VB and ILM proteins lead to an increase in eye size is unknown. The present study introduces the chick embryo as a model to study the role of ILM and VB in regulating eye size. The ILM and VB were disrupted by injecting collagenase into the eyes of E5 chick embryos. The digestion of VB and ILM proteins was monitored by Western blot and immunocytochemistry. Eye size was assessed up to 9 days after the enzyme injections. Intraocular injection of collagenase led to the disruption of the ILM and the VB by digesting their collagen constituents. Once disrupted, the ILM and the collagen II fibrillar network failed to regenerate despite continued synthesis of VB and ILM proteins. ILM and VB disruption resulted in eye enlargement of 50% within 4 days. The increase in eye size was greatly reduced by reconstituting the ILM. The present data show that the ILM and the VB play major roles in the early regulation of eye size. The authors speculate that the integrity of the vitreoretinal border is an important factor in preventing congenital high myopia.

A simple method for screening photoelectric dyes towards their use for retinal prostheses.

Photoelectric dyes absorb light and convert photon energy to electric potentials. To test whether these dyes could be used for retinal prostheses, a simple in vitro screening system was developed. Retinal neurons were cultured from the eyes of chick embryos at the 10-day embryonic stage, at which time no retinal photoreceptor cells have yet developed. Intracellular calcium elevation was observed with Fluo-4 in cultured retinal neurons before and after photoelectric dye was applied at varying concentrations to the culture medium. Five of 7 photoelectric dyes tested in this in vitro system induced intracellular calcium elevation in cultured chick retinal neurons. The intracellular calcium elevation generated by the 5 photoelectric dyes was blocked by extracellular calcium depletion in the case of all 5 dyes, and, except for one dye, by the presence of voltage-gated calcium channel blockers. The photoelectric dyes absorbed light under an inverted microscope and stimulated retinal neurons. This simple in vitro system allows the screening of photoelectric dyes which can be used for retinal prostheses.

Retinal growth hormone in the chick embryo.

The presence of GH and GH mRNA in the eyes of embryonic chicks is controversial and has, therefore, been further examined. In this study, GH cDNAs identical in size and sequence to the full-length pituitary transcript were generated by RT-PCR from mRNA extracted from the neural retinas of embryonic day (ED) 7 chick embryo eyes. GH immunoreactivity in the neural retina of embryos was primarily associated with proteins of 15 and 16 kDa, whereas only trace amounts of monomer (22-25 kDa) GH, the most abundant form in the pituitary gland, were present. GH immunoreactivity was also present in the vitreous humor, although this was associated only with the 15-kDa protein. After hatch, retinal proteins with GH immunoreactivity of 15 and 16 kDa were present neonatally but not after 42 d of age. The GH immunoreactivity in the neural retina of ED8 embryos was widespread, although GH staining was particularly abundant in retinal ganglion cells (RGCs). Full-length GH mRNA was similarly located, by in situ hybridization, throughout the neural retina and concentrated in cells in the RGC layer. The neural retina is also a site of GH action because 10-6 m chicken GH greatly increased (4- to 5-fold) the content of IGF-1 mRNA in 48-h cultured ED8 neural retinas. These results demonstrate the presence of full-length GH mRNA in the neural retina of chick embryos, in which GH immunoreactivity is primarily associated with RGCs and submonomer GH proteins of 15-16 kDa. These results also demonstrate GH action in the neural retina of embryos and suggest hitherto unsuspected roles for GH in retinal development and/or ocular function.

Lipid-mediated gene transfection into chick embryo retinal cells in ovo and in vitro

Several lipofection reagents were tested on chick embryo retinal cultures using green fluorescent protein (GFP) as a reporter gene; best results were obtained with the GenePORTER™ (GP) reagent, which yielded approximately 4.4% of the cells with intense GFP fluorescence. Cell survival and structural differentiation appeared normal, but one of the immunocytochemical markers studied (visinin) was less frequently observed in GP-treated cultures. When similar plasmid-GP mixtures were injected into chick embryo eyes in ovo, bright GFP-fluorescent cells were observed in different retinal layers, without detectable detrimental effects on retinal morphology. Particularly extensive reporter gene expression was obtained upon intraocular injection of GP plus naked DNA from a RCAS retrovirus, which resulted in the development of abundant radial columns of alkaline phosphatase-positive cells, separated by columns of negative cells. We conclude that lipid-based transfection offers a quick, simple and fairly innocuous means for gene delivery into proliferating and postmitotic retinal cells, in vitro as well as in the developing eye in ovo, and that transfection of naked retroviral DNA can lead to extensive expression of foreign genes by retinal cells, bypassing the time-consuming steps required for the generation of high-titer virion stocks.

Response of macrophage/microglial cells to experimental neuronal degeneration in the avian isthmo-optic nucleus during development.

Blockade of the retrograde axonal transport of isthmo-optic nucleus (ION) neurons in the avian embryo results in their massive degeneration. We used this system to investigate the response of macrophage/microglial cells to neuronal degeneration in the embryonic brain. Colchicine was injected into the right eye of quail or chick embryos at a time when the survival of ION neurons depends on retrograde trophic support from the retina, and the chronology of the subsequent macrophage/microglial response in the ION was analyzed. This response was restricted to the ION contralateral to the injected eye; no modifications of the normal state were observed in the surrounding parenchyma or in the opposite ION, used as control. The response was first detected 18 hours after the colchicine injection (18 hours pi), when an increase of the macrophage/microglial cell number was evident. The number of these cells in the affected ION increased, peaking at 40-48 hours pi. At later survival times, macrophage/microglial cells were progressively less abundant in the affected ION, which gradually diminished in size. At 120 hours pi the only remnant of the ION was a small cluster of macrophage/microglial cells, surrounded by a clear area with scarce nonmicroglial cells, in the region formerly occupied by the ION. This study reveals that a strong macrophage/microglial response occurs in the embryonic brain in response to neuronal degeneration but that these cells do not trigger the neuronal death, as they only appear after pyknotic fragments are already observable.

Connexin α1 and Cell Proliferation in the Developing Chick Retina

During the formation of the eye, high levels of connexin α1 (connexin 43) are expressed within the tissues of the cornea, lens, and neural retina. In order to determine whether connexin α1 plays a role in the regulation of cell proliferation we have used a novel antisense technique to reduce its expression early in development (embryonic days 2–4). Application of Pluronic gel, containing antisense oligodeoxynucleotides (ODNs) to connexin α1, to one eye of early chick embryos results in a rapid and significant reduction of α1 protein which lasts for 24–48 h. Embryos grown for 48 h, after ODN application to one eye, showed a marked reduction in the diameter of the treated, compared to that of the contralateral untreated, eye. Sections cut from the treated eyes showed that the retina was also reduced in size. TUNEL labeling and staining with propidium iodide showed that apoptosis within the retinae of both treated and untreated eyes was rare and thus that the reduction in the area of the retina brought about by antisense ODNs directed at connexin α1 was unlikely to be the result of increased cell death. However, the number of mitotic figures in the ventricular zone of the antisense-treated retinae revealed by propidium iodide staining was significantly reduced (P<0.0001) to 53 ± 3.5% (n=5) of that in the contralateral untreated control eyes. Embryos in which one eye was sham operated, treated with pluronic gel, or treated with sense ODN showed no significant changes in eye size or in the number of mitotic figures within the neural retina. These results point to a role for connexin α1-mediated gap-junctional communication in controlling the early wave of neurogenesis in the chick retina.

Disruption of the retinal basal lamina during early embryonic development leads to a retraction of vitreal end feet, an increased number of ganglion cells, and aberrant axonal outgrowth

Bacterial collagenase was injected into the vitreous of the eye of chick and quail embryos. Immunocytochemical and ultrastructural studies revealed that the collagenase dissolved the retinal basal lamina of the injected eye. The basal lamina disruption was first detectable 1 hour after enzyme injection and was complete within 3 hours. With further development, the retinal basal lamina was not reestablished; newly developing neuroepithelium in the peripheral retina, however, generated an intact basal lamina. Western blot analysis showed that Clostridial collagenase degraded various collagens but spared noncollagenous proteins. Basal lamina disruption of embryonic day 3 to 6 retinae led to the retraction of the end feet of the neuroepithelial cells, caused an increase in the number of Islet-1+ cells (most likely ganglion cells), an increase in the thickness of the optic fiber layer, and aberrant growth of optic axons on their way toward the optic disc. None of these changes were observed when retinal basal laminae were disrupted at later stages of development. The present data demonstrate that the retinal basal lamina, by anchoring the neuroepithelial cells to the pial surface of the retina, has an important function in the development of the normal cytoarchitecture of this structure. It is proposed that the altered extracellular environment in the vitreal part of the retina, resulting in the retraction of the neuroepithelial end feet, is responsible for the increased number of Islet-1+ cells and the aberrant axonal navigation.

Radio-iodination of neurotrophins and their delivery in vivo: advantages of membrane filtration and the use of disposable syringes

This paper reports two simple improvements for the radio-iodination of neurotrophins and their delivery in vivo. (1) Neurotrophins can be effectively separated from free iodide by using membrane filtration devices. Seven methods for the separation of free iodide are compared, including dialysis, gel filtration, and membrane filtration. Membrane filtration of the iodinated protein has several important advantages over dialysis or gel filtration. These include the precise control over the final concentration; excellent recovery of the neurotrophin; easy and inexpensive procedure; performance of the entire procedure in a fume hood; and reduced volume of radioactive waste. (2) Disposable, inexpensive syringes are suitable for the delivery of small volumes of radio-iodinated or non-radioactive neurotrophins. Plastic disposable insulin syringes are compared with Hamilton syringes. The ejection volume of the disposable syringes is surprisingly reliable in the dose range 2–15 μl. Their in vivo performance was tested by injections in the eyes of chick embryos in ovo. The amounts remaining in the eye varied significantly less with the disposable syringes. Leakage into the surrounding eye-muscles after intraocular injection was significantly more frequent with Hamilton syringes than with the disposable syringes. Thus, disposable syringes can be a reliable and cost-effective alternative for drug delivery of 2–15 μl volumes.

Gap Junctions between Mast Cells and Fibroblasts in the Developing Avian Eye

Mast cells are present in the eye of chick embryos from the 14th day onward, displaying metachromatic granules, mainly in the iris anterior surface and pectinate ligament. Ultrastructurally these cells show electron-dense granules and a few thin and short cytoplasmic projections in close contact with fibroblasts. Sometimes these contacts are extensive, with long fibroblast projections partially involving the mast cells. Gap junctions between mast cells and fibroblasts are observed only in the eyes of 16- and 20-day-old embryos. These intercellular specializations are represented by a close apposition of cytoplasmic membranes with an extension up to 300 nm. Gap junctions between mast cells and fibroblasts were not observed previously in vivo or in vitro, although in vitro studies have shown that a number of functionally critical interactions may occur between these cells. Our morphological findings suggest that, in vivo, fibroblasts interact with mast cells and may influence their maturation.

The structure of the developing chick retinal pigment epithelium revealed by high resolution scanning electron microscopy

The retinal pigment epithelium (RPE) in the developing eye of chick embryos has been studied during the early stages of development by high resolution scanning electron microscopy (HRSEM). Specimen preparation techniques which involve removal of the cytoplasmic matrix permitted visualization of organelles and other subcellular structures within RPE cells in detail and in three dimensional (3-D) stereo HRSEM. Using this technique, we were able to examine changes in melanosome structures during development and demonstrate that pigmentation in the RPE was present by day 4 of development. RPE plasma cell membranes showed extensive folding of the apical portion of the membrane closest to the developing neural retina by day 9. Examination of RPE photoreceptor junction revealed photoreceptor inner segments by day 6 and an outer segment by day 9. Mitochondria in the RPE were found to contain tubular cristae only. The ultra-structure in 3-D of the Golgi apparatus, smooth and rough endoplasmic reticulum, lysosomes and nuclear chromatin of the RPE, and Bruch's layer was revealed by the HRSEM method.

Reduction in the death and cytolamination of developing neurons by tetrodotoxin in the axonal target region

Tetrodotoxin was injected into one eye of chick embryos so as to block activity in the target territory of the isthmo-optic nucleus (ION) during its period of neuronal death. This markedly reduced the neuronal death and thereby prolonged the survival of some ‘aberrantly’ projecting neurons which would normally all die. In addition, the cytoarchitecture of the ION developed abnormally. Since these two effects differ markedly in their dose-dependence and in other ways, they cannot both be explained by changes in the strength of a single retrograde signal.

Cell death in the ventral region of the neural retina during the early development of the chick embryo eye.

The present study deals with morphologic and quantitative changes that take place in the area of cell death in the ventral part of the presumptive retinal wall of the chick embryo. These changes were followed from the optic vesicle stage until the first optic fiber fascicles leave the neural retina. Our results show that both the volume occupied by the area of cell death and the density of its pyknotic fragments undergo considerable variation during the period between Hamburger and Hamilton's (1951) stages 12 to 20. In the optic vesicle stages, cell death in the ventral wall of the vesicle was observed in 50 to 75% of the embryos studied. During stages 14 and 15, this zone was seen in more than 90%. By the time invagination of the optic cup was complete, the ventral retinal zone of cell death had disappeared entirely in a large proportion of embryos; in all others, it shrank significantly both in volume and density of pyknotic fragments. In stage 19, when the first optic fiber fascicles begin to emerge from the retina, a dramatic increase occurs in the number of pyknotic fragments in the posterior pole of the retina. The appearance of dying cells, in a region shortly to be traversed by developing ganglion cell axons, supports the hypothesis that cell death processes are apparently somehow related to the creation of a suitable environment for the emergence of fibers toward the optic stalk. Densities of mitotic and interphasic cells as well as the mitotic index were determined in both the retinal zone of cell death and in areas devoid of dead cells. In all developmental stages analyzed, the mitotic index was notably lower in the former than in non-necrotic zones, suggesting that cell proliferation is partially inhibited in retinal areas of cell death.

Development of the amino acid pools in chick embryo brain, heart, and eye: taurine, valine, glutamine, and phosphoethanolamine.

The redistribution of valine, from the nonrenewable yolk supply into excitable tissues, was studied during the first 15 days of chick embryogenesis. Valine levels in the extraembryonic circulation (the vitelline plexus) peak between days 7-9 (E7-9) and then decline steeply. In their first phase of differentiation (E2-E4), all embryonic tissues contain more valine than the blood plexus. From E4 to E7, the heart and brain exhibit initially a rapid fall in valine, but from day 7 on the decrease becomes more gradual. The eye during the same period reaches an equilibrium with circulating valine; as these levels fall from E9 to E15, the eye retains the valine that accumulated. Against this pattern of change, characteristic for an essential amino acid during embryogenesis, glutamine levels are at any time from two- to threefold higher than valine in all tissues. In the circulation, this ratio remains constant throughout the 15 days of embryonic development. Eye glutamine, higher on day 4, by E7 has entered into an equilibrium with glutamine in the plexus. A steady but two times higher glutamine level is maintained in the heart, although during the later stages of development it gradually tends to approach the plexus content. In sharp contrast, starting on E7 and accelerating on E9, a large increase of glutamine relative to valine or other essential amino acids is seen in developing brain tissue. This appears typical for most metabolic amino acids, suggesting that by days 9-10 the essential amino acid supply in the brain is being exhausted.(ABSTRACT TRUNCATED AT 250 WORDS)

Spatiotemporal gradients of kainate-sensitivity in the developing chicken retina.

We have studied the age-dependence of the effects of kainate (KA) on the chick retina as a prelude to the accompanying paper on the effects of target-removal on the isthmo-optic nucleus. KA was injected into the eyes of chick embryos and chicks at different ages, and the retinas were fixed a few hours or several days later. The former group of retinas was scanned for pyknotic cells. The earliest age at which KA caused pyknosis was embryonic day 10 (E10), when pyknotic cells appeared in a ventrotemporal patch in the amacrine sublayer near the fundus. Over the next two days the sensitive region expanded tangentially, reaching the periphery first temporally, then nasally. Only after E12 did the KA cause pyknotic cells to occur also in the bipolar sublayer, where the sensitivity spread in the same spatiotemporal sequence as the initial wave, but two days later. Cell loss was examined in embryos that survived a week or more after the KA injection. Substantial cell depletion was found in both the inner nuclear and ganglion cell layers, but only when the injection had been made after E12. With progressively later injections, the depleted zone expanded in the same spatiotemporal sequence as described above, until at E15 the injections caused depletion throughout the entire extent of the retina. The reasons for the lack of cell depletion after KA injections made before E12 are discussed. Cell counts in the ganglion cell layer and studies of anterograde transport of intravitreally injected peroxidase along the retinofugal fibers showed that about half the ganglion cells (including the displaced ganglion cells) pass through a period of vulnerability to the KA injections, to which they subsequently become sensitive.