The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods--proliferation, extracellular matrix maturation, and mineralization--and 2) two restriction points to which the cells can progress but cannot pass without further signals--the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle- and cell growth-regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.

Vertebrate hematopoietic stem cells are derived from ventral mesoderm, which is postulated to migrate to both extra-and intraembryonic positions during gastrula and neurula stages. Extraembryonic migration has previously been documented, but the origin and migration of intraembryonic hematopoietic cells have not been visualized. The zebrafish and most other teleosts do not form yolk sac blood islands during early embryogenesis, but instead hematopoiesis occurs solely in a dorsal location known as the intermediate cell mass (IM) of Oellacher. In this report, we have isolated cDNAs encoding zebrafish homologs of the hematopoietic transcription factors GATA-1 and GATA-2 and have used these markers to determine that the IM is formed from mesodermal cells in a posterior-lateral position on the yolk syncytial layer of the gastrula yolk sac. Surprisingly, cells of the IM then migrate anteriorly through most of the body length prior to the onset of active circulation and exit onto the yolk sac. These findings support a hypothesis in which the hematopoietic program of vertebrates is established by variations in homologous migration pathways of extra-and intraembryonic progenitors.

Normal rat osteoblasts in culture undergo a developmental sequence consisting of a proliferation period in which high levels of the histone and collagen type I genes are expressed, followed by periods of matrix maturation [high levels of alkaline phosphatase (AP)] and mineralization that signal a high level of production of osteopontin (OP) and osteocalcin (OC). Since these parameters are regulated by vitamin D, the effects of both short term and chronic treatment with 1,25-dihydroxyvitamin D3 were examined during osteoblast growth and differentiation. In acute studies, during the proliferation period, histone mRNA (reflecting DNA synthesis) was inhibited (20-60%). Matrix Gla protein (MGP) and OP mRNA were significantly elevated during proliferation (30- and 15-fold), in contrast to OC which is not expressed and was not induced by hormone treatment. OP and MGP remained stimulated throughout the developmental sequence, but to a lesser degree (from 6- to 10-fold). Collagen and AP mRNA were inhibited by hormone at their peak levels of expression, but were stimulated at their lowest basal levels in the mineralization period. OC expression, which was initiated at the onset of mineralization, was stimulated 13- to 15-fold when basal levels were low, then from 6- to 8-fold by hormone throughout its period of expression. In chronic studies a different profile of gene expression was observed. When hormone treatment was initiated during the proliferation period on day 6, type I collagen and AP expression were suppressed, mineralized nodules did not develop, and induced levels of OP and OC gene expression did not occur. When chronic treatment was initiated on day 20 after the development of a mineralized matrix, OC, but not collagen and OP, levels were stimulated by the hormone. This observation is consistent with the requirement of a competent or mineralized bone matrix for expression of OC. In contrast, MGP expression was stimulated in the chronic vitamin D-treated cultures similar to acute treatments. Taken together these studies demonstrate that vitamin D, a physiological mediator of bone formation and remodelling, can both positively and negatively regulate expression of osteoblast phenotypic markers as a function of duration of hormone treatment and basal levels of gene expression, which is a reflection of bone matrix competency and the differentiated state of the osteoblast.

Summary The medical records and radiographs of all horses with a third carpal bone fracture admitted to The Ohio State University Veterinary Hospital from 1979 to 1987 were reviewed. Three hundred and seventy‐one fractures were found in 313 horses; 57 per cent were Standardbreds, 41 per cent were Thoroughbreds, and only 1.6 per cent were Quarterhorses. All were young racehorses (average age = 3.1 years). Third carpal fractures occurred more frequently in the right limb (60 per cent) than the left limb (40 per cent); Thoroughbreds had a greater right‐left disparity (67.5 per cent R, 27.1 per cent L). Fractures were classified according to their size and anatomical location within the third carpal bone: incomplete fractures of the radial facet (type 1, N = 39), large proximal chip fractures of the radial facet (type 2, N = 140), small proximal chip fractures of the radial facet (type 3, N = 18), medial corner fractures (type 4, N = 13), frontal plane slab fractures of the radial facet (type 5, N = 93), large frontal plane slab fractures involving both the radial and intermediate facets (type 6, N = 35), fractures of the intermediate facet (type 7 N = 13), and sagittal slab fractures (type 8, N = 20). The incidence of each fracture type was significantly different between Standardbreds and Thoroughbreds. Type 1 and 2 fractures were more common in Standardbreds; type 5 and 6 fractures were more common in Thoroughbreds. Differences between these two breeds are related to the different gaits at which they race. The classification more accurately describes the extent of injury and the variation in fractures observed in this study than the traditional division as chips or slabs. A high quality skyline projection is important in correctly identifying these fractures; over 10 per cent of the fractures were detected only on this view.

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