The tesserate pattern of endoskeletal calcification has been investigated in jaws, gill arches, vertebral arches and fins of the sharks Carcharhinus menisorrah, Triaenodon obesus and Negaprion brevirostris by techniques of light and electron microscopy. Individual tesserae develop peripherally at the boundary between cartilage and perichondrium. An inner zone, the body, is composed of calcified cartilage containing viable chondrocytes separated by basophilic contour lines which have been called Liesegang waves or rings. The outer zone of tesserae, the cap, is composed of calcified tissue which appears to be produced by perichondrial fibroblasts more directly, i.e., without first differentiating as chondroblasts. Furthermore, the cap zone is penetrated by acidophilic Sharpey fibers of collagen. It is suggested that scleroblasts of the cap zone could be classified as osteoblasts. If so, the cap could be considered a thin veneer of bone atop the calcified cartilage of the body of a tessera. By scanning electron microscopy it was observed that outer and inner surfaces of tesserae differ in appearance. Calcospherites and hydroxyapatite crystals similar to those commonly seen on the surface of bone are present on the outer surface of the tessera adjacent to the perichondrium. On the inner surface adjoining hyaline cartilage, however, calcospherites of variable size are the predominant surface feature. Transmission electron microscopy shows calcification in close association with coarse collagen fibrils on the outer side of a tessera, but such fibrils are absent from the cartilaginous matrix along the under side of tesserae. Calcified cartilage as a tissue type in the endoskeleton of sharks is a primitive vertebrate characteristic. Calcification in the tesserate pattern occurring in modern Chondrichthyes may be derived from an ancestral pattern of a continuous bed of calcified cartilage underlying a layer of perichondral bone, as theorized by Ørvig (1951); or the tesserate pattern in these fish may itself be primitive.

The order of ossification of bones in the skeleton of Rana pipiens during larval growth and metamorphosis has been determined from observations on specimens fixed in 70% alcohol and stained with alizarin red S . The axial skeleton ossifies i n a generally cephalo-caudal sequence, beginning with the parasphenoid bone at Taylor-Kollros stages IV-IX, followed by vertebrae (V-IX) and then the urostyle (IX-XIV). Exoccipitals (VII-IX), frontoparietals (XI-XII) and prootics (XIII-XVII) are additional cranial bones which successively ossify before metamorphosis. With the onset of metamorphosis at stage XVIII jawbones and rostra1 bones of the skull ossify in the following succession: premaxilla, maxilla, septomaxilla, nasal, dentary, angular, squamosal, pterygoid, prevomer, mentomeckelian, quadratojugal, palatine, columella, posteromedial process of "hyoid." The sphenethmoid does not ossify until after metamorphosis.Ossification of limbbones begins with the femur or humerus at stages X-XI1 and progresses proximo-distally to the phalanges by stages XIII-XV. Carpals, however, do not ossify until stage XXV or after metamorphosis. The ilium of the pelvic girdle begins to ossify at stages X-XII, but the ischium is delayed until stages XX-XXIII. Scapula and coracoid of the pectoral girdle undergo initial ossification at stages XII-XIV, suprascapula and clavicle at stages XIII-XV. The sternum does not begin to ossify until stage XXIV. The possible role of thyroid hormones in stimulating osteogenesis is dicussed.

1. In the cytoplasm of oocytes of stage Y0, prior to the appearance of yolk, one observes a few scattered profiles of endoplasmic reticulum and numerous filamentous mitochondria, usually distributed at random but sometimes clustered. As the nuclear membrane begins to bulge outward, small granules and short rods appear in the perinuclear cytoplasm and endoplasmic reticulum becomes more prominent throughout the cytoplasm. 2. Coincident with the appearance of the first yolk platelets, which are deposited in a narrow peripheral ring within the endoplasm at stage Y1, protoplasmic processes, the microvilli, push out all over the surface of the oocyte. At the same time follicle cells pull away but remain attached to the oocyte at some points through finger-like processes which interdigitate with neighboring microvilli. It is estimated that the microvilli increase the absorptive area of the surface to about thirty-five times that of a simple sphere. Just beneath the microvillous layer is the basal protoplasm of the cortex, now containing tiny granules probably synthesized from newly absorbed raw materials. Cortical granules appear and become aligned below the basal layer on the external border of the endoplasm. Both the cortical granules and the yolk platelets measure up to 1 µ in diameter at this stage. 3. By stage Y3 (yolk filling peripheral three-fourths of cytoplasm), the basal layer of the cortex is folded so that it appears in section as alternating ridges and valleys. The microvilli now extend from the summits of the cortical ridges. Small, ring-shaped granules are abundant in the cortex. Cortical granules have increased to 2 µ in diameter. 4. Yolk platelets continue to be synthesized around the cortical granules and in the subjacent endoplasm. The largest platelets measured in the interior cytoplasm at stage Y4 (cytoplasm filled with yolk) were 3.7 µ wide by 5.8 µ long. Pigment granules increase in size from 0.15 µ in diameter at stage Y3 to 0.30 µ in diameter at stage Y4.

Tooth primordia at early stages of mineralization in the sharks Negaprion brevirostris and Triaenodon obesus were examined electron microscopically for evidence of ameloblastic secretion and its relation to calcification of the enamel (enameloid) layer. Ameloblasts are polarized with most of the mitochondria and all of the Golgi dictyosomes localized in the infranuclear end of the cell toward the squamous outer cells of the enamel organ. Endoplasmic reticular membranes and ribosomes are also abundant in this region. Ameloblastic vesicles bud from the Golgi membranes and evidently move through perinuclear and supranuclear zones to accumulate at the apical end of the cell. The vesicles secrete their contents through the apical cell membrane in merocrine fashion and appear to contribute precursor material both for the basal lamina and the enameline matrix. The enamel layer consists of four zones: a juxta-laminar zone containing newly polymerized mineralizing fibrils (tubules); a pre-enamel zone of assembly of matrix constituents; palisadal zones of mineralizing fibrils (tubules); and interpalisadal zones containing granular amorphous matrix, fine unit fibrils, and giant cross-banded fibers with a periodicity of 17.9 nm. It seems probable that amorphous, non-mineralizing fibrillar and mineralizing fibrillar constituents of the matrix are all products of ameloblastic secretion. Odontoblastic processes are tightly embedded in the matrix of the palisadal zones and do not appear to be secretory at the stages investigated. The shark tooth enamel layer is considered homologous with that of other vertebrates with respect to origin of its mineralizing fibrils from the innerental epithelium. The term enameloid is appropriate to connote the histological distinction that the enamel layer contains odontoblastic processes but should not signify that shark tooth enamel is a modified type of dentine. How amelogenins and/or enamelins secreted by amelo- blasts in the shark and other vertebrates are related to nucleation and growth of enamel crystallites is still not known.