Thrombospondin 1 is a secreted, trimeric glycoprotein that mediates interactions between cells and extracellular matrix and exhibits cell-specific effects on migration and proliferation. Recently, two additional thrombospondin genes (thrombospondin 2 and 3) have been identified. To study the functions of these proteins, we have used in situ hybridization and RNAse protection assays to compare the expression of the genes encoding thrombospondin 1, 2, and 3 during murine embryogenesis. Thrombospondin mRNAs were associated with ossification, neuronal organogenesis, and lung development, although transcripts were differentially expressed. Thrombospondin 1 was predominant from days 10 to 13. During this period, high but transient levels of expression were observed in the neural tube, head mesenchyme, and cardiac cushions. In contrast, a more constant level of thrombospondin 1 mRNA was apparent in resident megakaryocytes of the liver, as well as in circulating megakaryocytes; neither thrombospondin 2 nor 3 was detected in these cells. Thrombospondin 1 was also produced by cells of the developing kidney and gut. The expression of thrombospondin 2 was confined principally to organized connective tissue that included pericardium, pleura, perichondrium, periosteum, meninges, ligaments, and reticular dermis. Thrombospondin 2 was also produced by differentiating skeletal myoblasts and by cells of the kidney and gut. Moreover, high levels of expression were detected in blood vessels. Thrombospondin 3 mRNA was restricted to brain, cartilage, and lung. Although thrombospondin 1, 2, and 3 belong to a family of structurally related genes, the differences observed in the spatiotemporal distribution of the corresponding mRNAs indicate unique functions for these secreted proteins. 0 1993 Wiley-Liss, Inc.

Abstract. Sequences within the first intron of the od(I) collagen gene have been implicated in the regulation of expression of .l(I) collagen-reporter gene constructs in cultured cells. However, the physiological significance of these intronic elements has not been established. We have used in situ hybridization to examine whether a cell-specific pattern of expression of human oel (I) collagen-human growth hormone minigenes exists in transgenic mice. Our results indicate that transgenes which contained 2,300 bp of promoter/5' flanking sequence and an intact first intron were well expressed by fibroblasts in dermis and fascia, whereas transgenes lacking the intronic sequence, +292 to +1440, were not expressed in dermis and poorly expressed in fascia. Analysis of transgene expression in cultured fibroblasts obtained from dermal explants of transgenic animals confirmed the requirement for these intronic sequences in the regulation of the .l(1) collagen gene. In contrast, transgenes with or without the intronic deletion were expressed equally well in tendon and bone, in a manner comparable to the endogenous mouse od(I) collagen gene, and expression of neither transgene was detected in skeletal muscle or perichondrium. These data support a model in which cis-acting elements in the first intron, and their cognate DNA-binding proteins, mediate transcription of the al(I) collagen gene in some cells, such as dermal fibroblasts, but not in tendon cells or osteoblasts. Moreover, regions of the gene not included in the sequence, -2300 to +1440, appear to be required for transcription in tissues such as skeletal muscle and perichondrium.

Studies in vitro have not adequately resolved the role of intronic and upstream elements in regulating expression of the ftl(I) collagen gene. To address this issue, we generated 12 separate lines of transgenic mice with al(I) collagen-human growth hormone (hGH) constructs containing different amounts of 5'-flanking sequence, with or without most of the first intron. Transgenes driven by 2.3 kb of al(I) 5'-flanking sequence, whether or not they contained the first intron, were expressed at a high level and in a tissue-specific manner in seven out of seven independent lines of transgenic mice. In most tissues, the transgene was expressed at levels approaching that of the endogenous al(I) gene and was regulated identically with the endogenous gene as animals aged. However, in lung, expression of the transgene was anomalously high, and in muscle, expression was lower than that of the endogenous gene, suggesting that in these tissues other regions of the gene may participate in directing appropriate expression. Five lines of mice were generated containing transgenes driven by 0.44 kb of al(I) 5'-flanking sequence (with or without the first intron), and expression was detected in four out of five of these lines. The level of expression of the 0.44-kb constructs in the major collagen-producing tissues was 15-to 500-fold lower than that observed with the longer 2.3-kb promoter. While transgenes containing the 0.44-kb promoter and the first intron retained a modest degree of tissue-specific expression, those without the first intron lacked tissue specificity and were poorly expressed in all tissues except lung. These results contribute to our understanding of the role of the first intron in regulating ol(I) gene expression and identify a region, upstream of the basal al(I) promoter, which is necessary for full tissue-specific, developmentally regulated expression of the al(I) collagen gene.

The identification and functional analysis of DNA-protein interactions in the intronic and 5' flanking regions of the type I collagen genes has begun to define a series of cis-elements and trans-acting factors which regulate transcription of these genes. Studies such as these will eventually be expected to elucidate the mechanisms responsible for coordinate transcription of the alpha 1 and alpha 2 genes, a question which remains central to the field of collagen research. Although it is relatively straightforward to define sites of DNA-protein binding, interpretation of the functional importance of such interactions can be extremely complex. Furthermore, while mutation or deletion of a particular binding site may alter the functional activity of a construct transfected into cultured cells, there is no guarantee that a similar change will have the same effect in vivo, where the entire gene locus is present in its native chromosomal context. Nevertheless, these kinds of in vitro studies offer the best current approach to defining and isolating transcription factors that control expression of the alpha 1 and alpha 2 genes. Ultimately, it will be necessary to test the activity of such factors (and their respective cis-elements) in defined systems in vivo.

Although adequate hydration is essential for health, little attention has been paid to the effects of hydration among the generally healthy population. This narrative review presents the state of the science on the role of hydration in health in the general population, specifically in skin health, neurological function (i.e., cognition, mood, and headache), gastrointestinal and renal functions, and body weight and composition. There is a growing body of evidence that supports the importance of adequate hydration in maintaining proper health, especially with regard to cognition, kidney stone risk, and weight management. However, the evidence is largely associative and lacks consistency, and the number of randomized trials is limited. Additionally, there are major gaps in knowledge related to health outcomes due to small variations in hydration status, the influence of sex and sex hormones, and age, especially in older adults and children.