Age, 8.4 days.

Standard length, 3.57 mm.

Length of caudal fin, 0.30 mm. Mesencephalon width, 0.89 mm. Yolk depth, 1.45 mm.

The anatomy of this stage is described more fully than that of other stages for the reason that, in terms of organogenesis, de-velopment here may be considered completed. Descriptions of subsequent stages will be limited, for the most part, to gross mor phology and measurements.

A cephalic flexure is present here, with the telencephalon underlying the diencephalon in part (fig. 24A). The head is tightly enclosed within the pericardial extra-embryonic mem-branes (figs. 24B, 25A, B). The muscular and skeletal systems are well developed, and embryos of this stage, when removed from the ovarian follicles, swim about rapidly and will survive in conditioned aquarium water.

The telencephalon is short, and the lateral vesicles are solid and poorly distinguishable. The olfactory bulbs are solid masses of tissue attached lateroventrally to the telencephalon (fig. 25A). The diencephalon underlies the optic lobes of the mesencephalon (fig. 25B). At the level of the optic chiasma, a small middorsal extension of the diencephalon projects through the cleft between the optic lobes, i.e., the epiphysis (fig. 26A). The large hypothalamus extends ventrad and caudad to the level of the medulla (fig. 26B). The metencephalon is short, overlain in part by the optic lobes and in part by the myelen-cephalon.

Three layers of the sensory layer of the retina are visible, the innermost fibrous layer, a thick ganglionic layer, and the outermost nuclear layer (fig. 25B). A thin retinal pig-ment layer coats the sensory layer (fig. 25B). The choroid coat of the eye consists only of a pigment layer one or two cells in thickness (fig. 26B). The optic nerve leaves the retina from the posterior ventral quadrantand runs mesiad to form the optic chiasma (fig. 26A).

The otocyst consists of a large anterior vertical canal primordium, and smaller lateral and posterior vertical primordia. A prominent ventral saccular region and a dorsal medial saccus endolymphaticus are present (figs. 24A, 26A, B, 27A, B).

The mandibular arch possesses a set of small cartilages, most prominent of which are a pair of lateral primordia of the palato-quadrates with ventral extensions, i.e., Meckel’s cartilages (fig. 25B). The hyoid arch and in part the mandibular form the operculum enclosing four pairs of branchial arches (i.e., the third, fourth, fifth, and sixth visceral arches; figs. 24B, 27A, B, 28A, 32).

The opercula are separate at the midventral line from the level of the fifth visceral arch, where the ventral aorta enters the base of the gill arches. Each of the branchial arches is supplied by a large, unbranched aortic arch, and blunt, rounded gill filament primordia are present on the external surfaces of the gill bars (fig. 27A, B). In the posterior region of the pharynx, the roof, at the level of the third gill bar (fig. 27B), and the floor, at the level of the fourth gill and back (fig. 28A), possess rows of enamel organs which will form the pharyngeal teeth. Posterior to the fourth gill bar (sixth visceral arch), the pharynx possesses a pair of lateral evaginations which are presumed to be vestigial seventh visceral pouches (fig. 28B).

The esophagus is short, lined by cuboidal cells. This lining changes abruptly into a vacuolated columnar type at the level of the ductus pneumaticus (fig. 29B, C). The latter is a narrow duct with a fine lumen (fig. 30A). The basal portion of the swim bladder, where the duct enters, is lined by a high columnar epithelium which is greatly vacuolated and glandular in appearance (fig. 30A, B). The remainder of the swim bladder, extending to the right side and caudad almost as far as the anus, is retroperitoneal and possesses a squamous lining (figs. 30C, 3lA, 32).

The gall bladder projects ventrad and craniad in the mesentery mesiad to the liver (figs. 29C, 32). The exocrine portion of the pancreas may be found in irregular masses and lobules throughout the mesentery, scat-tered among the coils of the intestine, and embedded in the surface of the liver (figs. 29B, C, 30B, C, 32). The compact lobe of the pancreas is located in the mesentery at the level of the spleen, situated between the spleen and the liver (figs. 30A, 32). The cells of this lobe are tightly packed and possess large, darkly staining nuclei, basophilic cytoplasm, and small, slightly basophilic granules.

The intestine, with a small circular lumen and tall columnar epithelium, possesses a single loop extending craniad, ventrad, and somewhat to the right of the midline (figs. 29B, C, 30A, C, 31A-C, 32). The character of the epithelium does not change throughout its length. The anus, at the level of the sixth myomere, has a longitudinally oriented, slit-shaped opening (figs. 31 C, 32).

The anterior tip of the notochord is situated at a level just posterior to the auditory vesicle. The notochord is laterally compressed throughout its length (figs. 28A, 30C). Neurocranial cartilages are forming in the region ventral to the hind brain. Cartilage partially envelops the otocyst. Sclerotomal cartilages extend laterad from the notochord, forming the initial stages in the development of neural arches. The bases of the pectoral fins also contain cartilaginous plates.

The heart extends forward within the pericardial sac located under the head (figs. 24B, 25-27). The elongate sinus venosus drains the blood vessels of the yolk sac and the pericardial serosa. The atria1 region is poorly distinguished from the sinus. The ventricle protrudes to the right of the sinu- atrium. The conus arteriosus region is not distinguishable, and the long ventral aorta curves dorsally to enter the base of the gill arches at the level of the fourth branchial arch (sixth visceral arch). The ventral aorta turns anteriorly here and gives off, in the following order, the common bases for the fifth and sixth aortic arches, the fourth arch, the third arch, and the smaller second and first arches (fig. 33A, B). The median continuation of the ventral aorta forms the external carotid. The internal carotids originate from the third aortie arches. The third and fourth aortic arches enter the dorsal aorta separately from the fifth and sixth arches (fig. 33A, B). At this point, the mesonephroi surround the dorsal aorta, and numerous renal arteries are given off (fig. 28B). Caudad, a pair of large subclavian arteries are present, and a single, ventral coeliac artery (figs. 28B, 33A, B). The latter is relatively short, ramifying out to supply the viscera. The dorsal aorta continues caudad to become the caudal artery (figs. 31A, D, F, 33A, B).

The caudal veins drain the tail region (figs. 31D, E, F, G, 34A, B). The dorsal caudal vein runs along the dorsal aorta and in contact with it. The ventral caudal vein passes through the anal fin primordium (fig. 31G). The ventral caudal curves around craniad to the urinary bladder and runs ventrad to the right side of the intestine to form the vitello-caudal vein (figs. 3lA-D, 34A, B). The long, fine, posterior intestinal vein drains into this vessel (fig. 34B). This vessel eventually becomes part of the hepatic portal system, but retains its connection with the ventral caudal vein. Three vertical cross connections are present between the dorsal and ventral caudal veins. The anteriormost is at the level of the urinary pore (figs. 31D, 34B), the second at the level of the anal fin (fig. 31F), and the third at the base of the caudal fin. The dorsal caudal vein splits anteriorly to form two postcardinals (fig. 30B), the left one being much the smaller of the two (fig. 34A, B). The dorsal caudal and postcardinal veins actually form the renal portal system, draining into the numerous renal vessels. The subclavian veins and the anterior cardinal branches also drain into the renal plexus. Two large ducts of Cuvier col-lect the blood from the renal plexus (fig. 34A, B). A yolk sac portal sinus forms a ring around the trunk of the embryo, extending over the head as the posterior margin of the pericardial serosa. The vessel drains the vitellocaudal vein at its anal margin (figs. 31A, B, 34B). Laterally it drains the two ducts of Cuvier and a single hepatic vein entering the portal sinus on the left side (figs. 29A, B, 34A, B).

The hepatic portal vein forms a large sinus in the mesentery at the level of the spleen (figs. 30A, 34A, B).

The mesonephroi are limited to regions lateral to the notochord at the level of the base of the pectoral fin buds (fig. 28A, B). They are highly vascularized, partially enveloping the fork of the dorsal aorta. The mesonephric ducts become distinguishable near the caudal margins of the mesonephroi (fig. 29B). Here a small dorsal branch is given off by each of the ducts (fig. 29B, C). These accessory Wolffian ducts run caudad, closely appressed to the sides of the notochord. They possess a lumen, and an epithelium similar to that of the definitive ducts, and extend posteriorly to the level of the base of the swim bladder, where they end blindly.

The mesonephric (Wolffian) ducts con-tinue posteriorly, curving ventrad in the postanal region of the coelom, and empty into the base of a somewhat bilobed urinary bladder. The elongate urinary pore is situated posterior to the anus (fig. 31A, D, E).

The gonadial ridges become evident at-tached to the peritoneum undercoating the membranous portion of the swim bladder (fig. 30B, C). They extend caudad as far back as does the swim bladder (fig. 3lA, B). No evidence of sexual dimorphism in the gonads is visible at this stage.

The two lobes of the pituitary gland are completely separate. The infundibulum has moved back to a position posterior to the hypophysis and is poorly distinguishable from the hypothalamus. The hypophysis is detached from the stomodeal ectoderm and exhibits no differentiation into transitional, intermediate,, or anterior lobes (fig. 27A). A plexus of blood vessels derived from the “circle of Willis,” described for stage 12, supplies the pituitary. The epiphysis is small, saccate, and lodged between the optic lobes, and the connection with the diencephalon is narrowed down considerably (fig. 26A). The thyroid makes its first appearance in this stage or in stage 17. It is present as a diffuse group of small follicles around the ventral aorta at the level of the third and fourth aortic arches (figs. 26B, 27A). In later stages it becomes increasingly difficult to locate, as it migrates caudad in individual lobules or follicles.