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A fine structural investigation of the transport of intravenously-injected ferritin (110 A) through the external vascular barrier (choriocapillaris and Bruch's membrane) of the Japanese quail retina has been sequentially detailed. One minute after injection ferritin is found mainly within the vascular lumina of the choriocapillaris but also is seen in pinocytic vesicles within the capillary endothelium and dispersed throughout the various components of Bruch's membrane. Ferritin transport through the capillary endothelium appears to occur via pinocytic vesicles in the thickened portions and diaphragmatic pores (single and double) in the attenuated areas. Within 5–10 min post-injection ferritin is observed within the basal infoldings of the pigment epithelium.

The development of the cornea in the chick, based on the microscopical examination of serial sections of 108 morphologically staged chick embryos between stage 26 and hatching, is described. A description of the adult cornea is also provided. Correlations between the internal histogenesis of the cornea and the external morphology of the chick embryo have been established from the use of staged embryos, so that the probable state of development of the cornea can be assessed from surface appearances only. The cornea of the chick, at stage 26, consists of an anterior epithelium, an acellular postepithelial layer, and a mesothelium. Mesenchymal cells (corneal ‘corpuscles’) begin to infiltrate the postepithelial layer at stage 27. By stages 29–31 these cells occupy a continuous layer, the substantia propria, which comprises the entire extent of the postepithelial layer except for an anterior, acellular band subjacent to the anterior epithelium. The anterior band becomes progressively thinner and disappears at stage 36, the substantia propria then extending across the entire distance between the anterior epithelium and the mesothelium. The anterior limiting lamina first appears at stage 39. It closely resembles and is often confused with the temporary anterior band of the postepithelial layer. The posterior limiting lamina was not identified with certainty in any of the embryonic corneae examined. It has been stressed that embryological studies in which external staging and internal development are correlated provide a more precise interpretation of developmental processes, and are an essential prelude to experimental, physiological, biochemical, and histochemical investigations of embryonic development.

The practicability of applying histochemical reactions to bulk staining has been explored by subjecting whole chick embryos at early stages to the periodic acid-Schiff (PAS) reaction. A comparison of the microscopic distribution of PAS positive substances revealed by this procedure with that obtained by the standard routine, i.e., staining of deparaffinized sections on slides, has shown similar localizations of PAS positive material and, in addition, finer morphological detail and more intensive reactions by staining the specimens in toto. The following method is recommended for chick embryos between stages 11-17 (Hamburger and Hamilton): Fixation in Gendre's fluid at 4°C; oxidation with alcoholic buffered periodic acid, 15 min; rinsing in distilled water, 10 min; Schift's reagent, 30 min; 3 sulfite rinses, 5 min each; running tap water, 10 min; dehydration, clearing and double-embedding in celloidin and paraffin.

The intraembryonic distribution of selectively stained (periodic acid-Schiff positive) primordial germ cells (PGCs) was investigated in morphologically staged chick embryos from the stage when they first arrive in the embryo proper until they have become firmly established in the gonadal anlage. The number and precise localization of the PGCs at each developmental stage have been recorded and have provided significant data on the mechanisms involved in the transport and disposition of the PGCs, particularly their colonization of the gonadal primordium. In general, the PGCs of the chick measure between 12 and 14 μ in diameter and possess abundant intracytoplasmic deposits of PAS-positive glycogen. They originate extraembryonically and are transported passively by the circulating blood to all vascularized parts of the developing embryo. The time of their first appearance within the embryo proper (stage 12) coincides with the onset of cardiac propulsion and blood circulation. Thereafter, the number of PGCs increases steadily from an average of 30 at stage 13 to an average of 894 at stage 17. In all stages examined the PGCs are evenly distributed on the right and left sides of the body. Initially, the PGCs are observed throughout the existing vascular channels, particularly in the heart, great vessels, and the small vessels of the cephalic mesenchyme. Many are found in such incongruous sites as the notochord, neural and surface ectoderm, and endoderm. By stage 15, however, the majority of PGCs are concentrated in the bilateral future gonadal region, i.e., an extensive longitudinal area of mesodermal tissue surrounding the medial portion of the intraembryonic coelom caudal to the place of exit of the omphalomesenteric arteries. The pattern of distribution within the gonadal territory varies with the developmental stage and appears to be determined by the morphological and concomitant vascular changes which occur there. At stage 15, for example, the dorsal aorta is situated directly medial to the medial angle of the gonadal territory and gives off splanchnopleuric branches which form a capillary network below the floor of the coelom. Because this is the major blood supply of the gonadal region at this stage, the vast majority (98%) of the intragonadal PGCs are confined to the capillary stroma and the epithelium of this zone. The small caliber of the capillaries compared to the large size of the PGCs serves to impede circulation here so that the PGCs become halted and then are able to begin their active amoeboid movement and to invade the overlying coelomic epithelium. Subsequent medial displacement of the coelomic cavity and aorta permits a gradual overlapping of the roof of the coelom by the dorsal aorta and concomitant alterations in the vascular relationships so that by stage 18 the majority of PGCs come to occupy the medial angle and roof zones, either by migrating from the floor region or by actively penetrating the aortic wall.

The relative proportion of rods and cones (single and double) in the chicken retina was determined by counting these elements in each of nine pre-determined segments prepared as periodic acid-Schiff serial sections for rod (hyperboloid glycogen) and double cone (accessory cone paraboloid glycogen) identifications and as unfixed, flat preparations for cone determinations (colored oil droplets). Periodic acid-Schiff preparations revealed that rods and accessory cones were equal in number in all retinal segments except those located directly posterior to the posterior extremity of the pecten where the double cones out-numbered the rods in a ratio of 14 : 10. On the basis of oil-droplet pigmentation, double cones housing a golden-yellow droplet in their chief component and a small yellowish-green droplet in their accessory members were constantly observed to be twice as numerous as single cones (red droplet) in all retinal segments. These data reveal that the proportion of rods to double and single cones is 2 : 2 : 1, respectively, in all areas of the chicken retina except a posterior (lateral) segment where the ratio is 10 : 14 : 7. This area of greater cone concentration corresponds to the site of the temporal fovea of some birds and may represent the area of most acute vision in the fovea-free chicken retina.

Phase microscopic investigations of Kolmer-fixed, depigmented sections of the adult chicken retina have provided photomicrographic evidence of the existence of three different photoreceptors: single rods, single cones, and double cones. The rod extends the entire thickness of the visual cell layer and is characterized by a uniformly thick outer segment and a hyperboloid-containing inner segment which is devoid of an oil droplet. The single cone is the shortest element; it contains a red oil droplet. The double cone consists of two unequal members, a tall, slender chief cone and a broad accessory cone. The chief component contains a large yellow oil droplet, whereas the accessory cone houses a small, oval, yellowish-green droplet and a characteristically large, oval paraboloid. The rod hyperboloid and the accessory cone paraboloid contain glycogen. No colorless droplets have been observed. Owing to the close association between oil droplet color and cone type, three colored layers of oil droplets are formed within the thickness of the retina: a proximal row of red droplets (the short, single cones), an intermediate layer of yellowish-green droplets (the accessory cones), and a distal row of yellow droplets (the tall chief cones).

1. 1. The feasibility of producing colonies of Japanese quail ( Coturnix coturnix japonica ) with carotenoid-free (“colorless”) retinal oil droplets has been demonstrated by dietary exclusion of carotenoids. 2. 2. Adult quail maintained on carotenoid-free rations undergo a gradual reduction in blood and liver carotenoid level; their “colorless” offspring possess normal growth and reproductive characteristics as well as unaltered visual cell morphology. 3. 3. Zeaxanthin is the major carotenoid found in quail blood. 4. 4. Hydrolysis of quail retinal extracts yields zeaxanthin and astacene. The latter is derived from astaxanthin and/or its esters.

The uItrastructure oft he pigment epithelium and its vascular barrier was examined in the Japanese quail by electron microscopy. Most endothelial pores in the choriocapillaris appear bridged by double diaphragms. The pigment epithelium is characterized by numerous slender basal infoldings, myeloid bodies and phagosomes. Myeloid bodies communicate with the nuclear envelope and profiles of both the rough and smooth endoplasmic reticulum. Phagosome formation appears to be accomplished by concomitant activity of the rod itself (curling of apical lamellae) and the apical villi of the pigment epithelium. Within the pigment epithelium cytoplasm the phagosomes undergo degeneration and are associated with increased numbers of lysosomal-like granules.

The feasibility of studying avian primordial germ cells in blood smears has been demonstrated. Blood smears prepared from chick embryos of stages 13 to 15 (48 to 55 hours) contained primordial germ cells, which were revealed by the periodic acidSchiff reaction. The presence of glycogen in the cytoplasm of the primordial germ cells facilitated their selective identification.

The origin of the colored oil droplets in the chick cones was investigated by subjecting retinas from staged embryos to correlative biochemical (visible spectrophotometric assays of chromatographically separated extracts) and microscopic analyses. The carotenoid pigments responsible for the coloration of the oil droplets are detected spectrophotometrically before they can be visualized microscopically in the form of colored droplets. The spectrophotometric data reveal that each pigment arises independently in the following sequence: golden yellow, yellowish green, and red. The golden yellow hydrocarbon is first detected at stage 27 at which time the retina consists of a thick, unstratified, neuroepithelial layer with an active proliferative zone, but lacks visual cells. Galloxanthin, the yellowish green pigment, arises at stage 34. The retina is still devoid of visual elements at this stage but is organized into several nuclear layers. The concentrations of the golden yellow and yellowish green carotenoids begin to increase rapidly at stage 37 when inner segments are differentiating, but oil droplet vacuoles have not yet appeared within them. Colorless droplets first evident in fresh preparations of this stage may not be colorless at all, but may contain the yellow pigments in sach low concentrations that their colors cannot be discriminated microscopically. The red pigment, astaxanthin, is first evident at stage 41 and immediately exhibits a steady increase in concentration. The retina possesses adult stratification at this time; all ten layers are present and oil droplet vacuoles are observed for the first time. On the basis of the spectrophotometric findings it is contended that the double cones are the first cones to differentiate in the chick retina and that the chief component (bearer of the golden yellow droplet) arises before the accessory element (carrier of the yellowish green droplet).