The elaboration of dentin collagen precursors by the odontoblasts in the incisor teeth of 30-40-g rats was investigated by electron microscopy, histochemistry, and radioautography after intravenous injection of tritium-labeled proline .At 2 min after injection, when the labeling of blood proline was high, radioactivity was restricted to the rough endoplasmic reticulum, indicating that it is the site of synthesis of the polypeptide precursors of collagen, the pro-alpha chains .At 10 min, when the labeling of blood proline had already declined, radioactivity was observed in spherical portions of Golgi saccules containing entangled threads, and, at 20 min, radioactivity appeared in cylindrical portions containing aggregates of parallel threads . The parallel threads measured 280-350 nm in length and stained with the low pHphosphotungstic acid technique for carbohydrate and with the silver methenamine technique for aldehydes (as did extracellular collagen fibrils) . The passage of label from spherical to cylindrical Golgi portions is associated with the reorganization of entangled into parallel threads, which is interpreted as the packing of procollagen molecules .Between 20 and 30 min, prosecretory and secretory granules respectively became labeled . These results indicate that the cylindrical portions of Golgi saccules transform into prosecretory and subsequently into secretory granules . Within these granules, the parallel threads, believed to be procollagen molecules, are transported to the odontoblast process .At 90 min and 4 h after injection, label was present in predentin, indicating that the labeled content of secretory granules had been released into predentin . This occurred by exocytosis as evidenced by the presence of secretory granules in fusion with the plasmalemma of the odontoblast process .It is proposed that pro-alpha chains give rise to procollagen molecules which assemble into parallel aggregates in the Golgi apparatus . Procollagen molecules are then transported within secretory granules to the odontoblast process and released by exocytosis . In predentin procollagen molecules would give rise to tropocollagen molecules, which would then polymerize into collagen fibrils .

A peptide that is rich in organically bound phosphorus and contains abundant serine residues has been identified in the dentin of man (1), fetal bovine (2, 3), and rat (4). This phosphoprotein may play a role in mineralization (5-9). Butler et al. (4) reported that the phosphoprotein of rat incisor dentin constituted 10.8% of the proteinaceous material recovered from decalcified incisor teeth while collagen comprised 84%. Since the phosphorus content of the phosphoprotein was estimated at 3.29% and that of collagen at 0.19% (4), much of the phosphorus taken up in organic form by the incisor would likely be present as phosphoprotein. With this in mind, it was decided to inject [~3P]phosphate into rats and examine the demineralized incisor teeth by radioautography in the hope of tracing phosphoprotein formation.The organic phosphorus of dentin phosphoprotein is believed to be attached to serine residues (6). In the rat incisor dentin, this amino acid comprises 35 residues per cent of the phosphoprotein and only four residues per cent of the cyanogen bromide peptides of collagen (4). Hence serine also appeared to be a suitable amino acid precursor to trace phosphoprotein formation by radioautography.Finally, the radioautographic pattern of the deposition of labeled phosphorus and serine was compared to that of [3H]proline. Proline may be used as a precursor to trace collagen, since it makes up 22.0 residues per cent of dentin collagen and only 2.4 residues per cent of the phosphoprotein (4). The results indicated that the pattern of phosphoprotein deposition into the dentin matrix is strikingly different from that of collagen. The fixative consisted of 2.5% glutaraldehyde in 0.05 M S6rensen's phosphate buffer with the addition of 0.1% sucrose and 0.5% dextrose. In the experiments using L-[2,~-gH]proline, the fixative employed was 3% formaldehyde (TAAB Laboratories, Emmer Green, Reading, England) in 0.1 M S6ren-sen's phosphate buffer with 0.1% sucrose added. The final pH of either fixative was 7.2-7.3. After perfusion for 15 min at room temperature the maxillary incisor teeth were immersed in fresh fixative for 2-3 h and demineralized in EDTA for 2 wk at 4°C (10). Although a 2-wk period is sufficient for exhaustive demineralization, some teeth were denfineralized for 3 or 4 wk in the experiments conducted with [3~P]phosphate. Specimens were washed overnight in 0.15 M S6rensen's buffer, sliced transversely with razor blades into 1-mm thick sections, postfixed for 1-2 h in 1% OsO4 in 0.1 IV[ S6rensen's buffer, dehy-838 MATERIALS AND METHODS Sherman

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The differentiation of the brush border which makes up the apical free surface of intestinal absorptive cells has been studied by electron microscopy . Specimens of Xenopus small intestine were fixed at various stages during metamorphosis, the time when a new intestinal epithelium forms . The interpretation of details described herein emphasizes the role of "surface-forming" vesicles . These vesicles are thought to provide membrane both for the initial expansion of the apical surface and for the later elongation of the microvilli . The latter are believed to be "molded" around filamentous cores that appear early in differentiation . The cores are attached to the apical membrane and extend vertically into the supranuclear cytoplasm . This interpretation rests chiefly on (a) the resemblance, both in morphology and in staining properties with colloidal thorium, between the membrane that limits the vesicles and that which limits the microvilli and (b) the distribution and time of appearance of the vesicles with respect to development of the microvilli . According to this view, the specific properties of surface membrane reside in preformed units that arise within the supranuclear cytoplasm. This morphogenetic process probably involves participation of the Golgi region as the site where the complex macromolecular architecture of the cell surface is assembled .

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