Regulated expression of proenkephalin A in normal lymphocytes

The expression of proenkephalin A (PEA), a neuropeptide-encoding gene, was examined in normal rat lymphocytes. With the use of Northern blot hybridization analysis of total RNA, PEA mRNA was found in normal cells derived from spleen, lymph nodes, and bone marrow. Cell sorting of the two main fractions of B and T cells derived from the spleen revealed that PEA is expressed in normal B cells (sIg+). The expression of PEA mRNA was markedly enhanced after a short incubation (3 h) of cells with LPS or Salmonella typhimurium. This was not the case when these cells were incubated with Con A during the same period of time; whereas, in thymocytes the presence of PEA mRNA was exclusively dependent upon mitogenic stimulus (Con A) and could be detected after 24 h of in vitro incubation. Extracts of cells were also found to contain immune reactive enkephalins, indicating that the PEA mRNA is translated. These results support the concept that neuropeptides, such as enkephalins, have a role in the modulation of the immune response and may participate in the bidirectional communication between the nervous and immune systems.     

Regulation of proenkephalin expression in cultured skin mesenchymal cells.

Proenkephalin, a classically defined opioid encoding gene, is transiently expressed in nondifferentiated mesodermal cells during organogenesis. We examined the hypothesis that this expression is associated with mesenchymal cell proliferation. For this purpose, we established a cell culture derived from fetal skin mesenchyme that specifically expresses proenkephalin mRNA in correlation with hypodermis development. These mesenchymal cells also produce and secrete significant amounts of proenkephalin-derived peptides. Using this model system, we observed a marked increase in proenkephalin mRNA expression in response to serum. This effect is time dependent and reaches peak levels during the G1/S transition. Similarly, 12-O-tetradecanoyl-phorbol-13-ester, whose biological actions have been shown to be mediated by the activity of protein kinase C (PKC), up-regulates proenkephalin expression. Desensitization of PKC by prolonged exposure of cells to 12-O-tetradecanoyl-phorbol-13-ester attenuates the serum induction of proenkephalin. The results presented in this report demonstrate that proenkephalin expression in mesenchymal cells is regulated by serum factors via mechanisms that involve PKC activity. A possible association between proenkephalin expression and cell proliferation is suggested.     

Regulated gene expression of hyaluronan synthases during Xenopus laevis development

Here reported is the developmental gene expression pattern of the three known vertebrate hyaluronan synthases (XHas1, XHas2 and XHas3) and a comparative analysis of their mRNAs spatio-temporal distribution during Xenopus laevis development. We found that while XHas2 shows a steady-state expression from gastrula to late tailbud stage, XHas1 is mainly present in the early phases of development while XHas3 is predominantly transcribed in tailbud embryos. XHas1, XHas2 and XHas3 show distinct tissue expression patterns. In particular, XHas1 is localized in ectodermal derivatives and in cranial neural crest cells, whereas XHas2 is mainly found in mesoderm-derived structures and in trunk neural crest cells. Moreover, the expression pattern of XHas2 overlaps that of MyoD in cells committed to a muscle fate. Unlike the other hyaluronan synthases, XHas3 mRNA distribution is very restricted. In particular, XHas3 is expressed in the otic vesicles and closely follows the inner ear development. In conclusion, XHas1, XHas2 and XHas3 mRNAs have distinct and never overlapping spatial expression domains, which would suggest that these three enzymes may play different roles during embryogenesis.     

Regulated expression of a cell division control-related protein kinase during development

Protein kinases are important signaling molecules that are known constituents of cellular pathways critical for normal cellular growth and development. We have recently identified a new protein kinase, p58, which contains a large domain that is highly homologous to the cell division control p34cdc2 protein kinase. This new cell division control-related protein kinase was originally identified as a component of semipurified galactosyltransferase; thus, it has been denoted galactosyltransferase-associated protein kinase. In vitro, this protein kinase has been shown to phosphorylate a number of substrates, including histone H1, casein, and galactosyltransferase. In vivo, we have found that this protein kinase affects galactosyltransferase enzyme activity and that it is apparently involved in some aspect of normal cell cycle regulation. In this report, we find that the p58 gene is evolutionarily well conserved and expressed ubiquitously, but to varying extents, in adult tissues. In developmentally staged embryos, p58 expression was elevated early in embryogenesis and then decreased dramatically. In the murine submandibular gland, p58 expression was elevated between day 14 and day 16 post coitus. Expression in the submandibular gland appeared to parallel the proliferation and differentiation of specific cell types as judged by in situ hybridization. These studies indicate that the p58 protein kinase may have a critical function during normal embryonic development and that this protein kinase continues to be expressed in differentiated adult tissues.     

Expression of protein kinase C genes during ontogenic development of the central nervous system.

We have analyzed the RNA expression of three protein kinase C (PKC) genes (alpha, beta, and gamma) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)+ RNA indicates that expression of PKC alpha and beta genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage, and that the PKC gamma gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)+ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenetic patterns were similar to those observed for human brain. Furthermore, we observed that the expression of PKC gamma is induced in the peri- and postnatal phases. These results suggest that expression of PKC alpha, beta, and gamma genes possibly mediates the development of central neuronal functions, and expression of PKC gamma in particular may be involved in the development of peri- and postnatal functions.     

Regulated expression of ADPglucose pyrophosphorylase and chalcone synthase during root development in sweet potato

The phase transition in sweet potato root during tuber differentiationis a complex developmental process that involves changes in gene expression andmorphogenesis. Among the three kinds of root in sweet potato (white fibrousroot, thick-pigmented root and lateral root), ADP-glucose pyrophosphorylase(AGPase) and chalcone synthases (CHS) are expressed only in thick-pigmentedroots after 3 weeks, and this also depends on the developmental stage. Sinceexposing roots to the light or culturing under hydroponic conditions inhibitstuber formation in sweet potato, the expression of AGPase and CHS was studiedunder light and dark conditions. AGPase and CHS expression in sweet potatorootswas suppressed very sensitively by light or water stress, similar to rootdevelopmental patterns. Based on an analysis of AGPase and CHS expression indifferent kinds of root tissues and in different developmental stages, thesegenes were shown to be closely associated with the differentiation ofthickeningpigmented roots.     

Regulated temporal and spatial expression of the calcium-binding proteins calcyclin and OPN (osteopontin) in mouse tissues during pregnancy.

In situ hybridization and northern/slot blot analyses were used to quantify the expression of calcyclin (2A9, 5B10), osteopontin (opn, secreted phosphoprotein, 2ar) and calmodulin mRNAs in mouse tissues that support pregnancy. High-to-moderate levels of the mRNAs of all three genes were detected at discrete locations in the uterus, decidua and placenta as a function of gestation time. Calmodulin expression was constant in these tissues; calcyclin mRNA was high during early pregnancy and declined after day 8-9 of gestation; and opn mRNA was undetectable before day 7, with maximal levels on days 9-12 in each of these tissues. Calcyclin, but not opn, expression was also observed in the chorioamnion after day 12. Calcyclin was expressed throughout the decidua on day 8 but became restricted to the primary (antimesometrial) decidual zone and decidua lateralis on day 9, and the decidua capsularis after day 9. By contrast, opn mRNA was localized on day 9 to the mesometrial triangle, which contains a large population of granulated metrial gland cells, and to the decidua basalis. These two genes may serve as markers for the two types of decidual tissue. We suggest that one function of OPN, which may be an indicator of cells in the decidua that have a bone marrow genealogy, is to mediate the flux of calcium from the maternal circulation to the developing embryo.     

Characteristic tetraspanin expression patterns mark various tissues during early Xenopus development

The tetraspanins (Tspans) constitute a family of cell surface proteins with four transmembrane domains. Tspans have been found on the plasma membrane and on exosomes of various organelles. Reports on the function of Tspans during the early development of Xenopus have mainly focused on the expression of uroplakins in gametes. Although the roles of extracellular vesicles (EVs) including exosomes have been actively analyzed in cancer research, the contribution of EVs to early development is not well understood. This is because the diffusivity of EVs is not compatible with a very strict developmental process. In this study, we analyzed members of the Tspan family in early development of Xenopus. Expression was prominent in specific organs such as the notochord, eye, cranial neural crest cells (CNCs), trunk neural crest cells, placodes, and somites. We overexpressed several combinations of Tspans in CNCs in vitro and in vivo. Changing the partner changed the distribution of fluorescent-labeled Tspans. Therefore, it is suggested that expression of multiple Tspans in a particular tissue might produce heterogeneity of intercellular communication, which has not yet been recognized.     

Telokin expression is restricted to smooth muscle tissues during mouse development

Telokin is a 17-kDa protein with an amino acid sequence that is identical to the COOH terminus of the 130-kDa myosin light chain kinase (MLCK). Telokin mRNA is transcribed from a second promoter, located within an intron, in the 3' region of the MLCK gene. In the current study, we show by in situ mRNA hybridization that telokin mRNA is restricted to the smooth muscle cell layers within adult smooth muscle tissues. In situ mRNA analysis of mouse embryos also revealed that telokin expression is restricted to smooth muscle tissues during embryonic development. Telokin mRNA expression was first detected in mouse gut at embryonic day 11.5; no telokin expression was detected in embryonic cardiac or skeletal muscle. Expression of telokin was also found to be regulated during postnatal development of the male and female reproductive tracts. In both uterus and vas deferens, telokin protein expression greatly increased between days 7 and 14 of postnatal development. The increase in telokin expression correlated with an increase in the expression of several other smooth muscle-restricted proteins, including smooth muscle myosin and alpha-actin.     

Taurine distribution in rat tissues during development.

1. Taurine levels have been determined in eight rat organs. 2. During postnatal growth the taurine content in retina, heart, small intestine, spleen and lung increases with advancing age, although adult values are not reached at the same time. 3. In contrast the taurine content decreases with age in brain cortex, liver and kidney. 4. The taurine in subcellular fractions of adult, 20-day-old and 5-day-old rat tissues exists predominantly in the cytosol of the cell. Taurine content in particulate fractions shows marked variations during development in the different organs. 5. Taurine distribution in the subcellular fractions suggests that some of the cellular taurine in the tissues is not freely mobile in cytosol.     

Different patterns of gene expression of topoisomerase II isoforms in differentiated tissues during murine development.

The expression of DNA topoisomerase II alpha and beta genes was studied in murine normal tissues. Northern blot analysis using probes specific for the two genes showed that the patterns of expression were different among 22 tissues of adult mice. Expression levels of topoisomerase II alpha gene were high in proliferating tissues, such as bone marrow and spleen, and undetectable or low in 17 other tissues. In contrast, high or intermediate expression of topoisomerase II beta gene was found in a variety of tissues (15) of adult mice, including those with no proliferating cells. Topoisomerase II gene expression was also studied during murine development. In whole embryos both genes were expressed at higher levels in early than late stages of embryogenesis. Heart, brain and liver of embryos two days before delivery, and these same tissues plus lung and thymus of newborn (1-day-old) mice expressed appreciable levels of the two genes. Interestingly, a post-natal induction of the beta gene expression was observed in the brain but not in the liver; conversely, the expression of the alpha gene was increased 1 day after birth in the liver but not in the brain. However, gene expression of a proliferation-associated enzyme, thymidylate synthase, was similar in these tissues between embryos and newborns. Thus, the two genes were differentially regulated in the post-natal period, and a tissue-specific role may be suggested for the two isoenzymes in the development of differentiated tissues such as the brain and liver. Based on the differential patterns of expression of the two isoforms, this analysis indicates that topoisomerase II alpha may be a specific marker of cell proliferation, whereas topoisomerase II beta may be implicated in functions of DNA metabolism other than replication.     

Distinct expression of type XIII collagen in neuronal structures and other tissues during mouse development.

Type XIII collagen is a type II transmembrane protein found in adhesive structures of mature tissues. We describe here its expression and spatio-temporal localization during mouse fetal development. Type XIII collagen mRNAs were expressed at a constant rate during development, with an increase of expression towards birth. Strong type XIII collagen expression was detected in the central and peripheral nervous systems of the developing mouse fetus in mid-gestation. Cultured primary neurons also expressed this collagen, and it was found to enhance neurite outgrowth. The results suggest that type XIII collagen is a new member among the proteins involved in nervous system development. Strong expression during early development was also detected in the heart, with localization to cell-cell contacts and accentuation in the intercalated discs perinatally. During late fetal development, type XIII collagen was observed in many tissues, including cartilage, bone, skeletal muscle, lung, intestine and skin. Clear developmental shifts in expression suggest a role in endochondral ossification of bone and the branching morphogenesis in the lung. Notable structures lacking type XIII collagen were the endothelia of most blood vessels and the endocardium. Its initially unique staining pattern began to concentrate in the same adhesive structures where it exists in adult tissues, and started to resemble that of the beta1 integrin subunit and vinculin during late intrauterine development and in the perinatal period.     

NUDC expression during amphibian development.

To identify gene products important for gastrulation in the amphibian Pleurodeles waltl, a screen for regional differences in new protein expression at the early gastrula stage was performed. A 45 kDa protein whose synthesis was specific for progenitor endodermal cells was identified. Microsequencing and cDNA cloning showed that P45 is highly homologous to rat NUDC, a protein suggested to play a role in nuclear migration. Although PNUDC can be detected in all regions of the embryo, its de novo synthesis is tightly regulated spatially and temporally throughout oogenesis and embryonic development. New PNUDC synthesis in the progenitor endodermal cells depends on induction by the mesodermal cells in the gastrula. During development, PNUDC is localized in the egg cortical cytoplasm, at the cleavage furrow during the first embryonic division, around the nuclei and cortical regions of bottle cells in the gastrula, and at the basal region of polarized tissues in the developing embryo. These results show for the first time the expression and compartmentalization of PNUDC at distinct stages during amphibian development.     

Cathepsin expression during skeletal development.

Cysteine proteinases, cathepsins B, H, K, L and S, have been implicated in several proteolytic processes during development, growth, remodeling and aging, as well as in a variety of pathological processes. For systematic analysis of cathepsin gene expression we have produced cDNA clones for mouse and human cysteine cathepsins. Northern analysis of a panel of total RNAs isolated from 16-19 different human and mouse tissues revealed the presence of mRNAs for cathepsin B, H, K, L and S in most tissues, but each with a distinct profile. Of the different cathepsin mRNAs, those for cathepsin K were clearly the highest in bone and cartilage. However, relatively high mRNA levels for the other cathepsins were also present in these tissues. To better understand the roles of different cathepsins during endochondral ossification in mouse long bones, cathepsin mRNAs were localized by in situ hybridization. Cathepsin K mRNAs were predominantly seen in multinucleated chondroclastic and osteoclastic cells at the osteochondral junction and on the surface of bone spicules. The other cathepsin mRNAs were also seen in osteoclasts, and in hypertrophic and proliferating chondrocytes. These observations were confirmed by immunohistochemistry and suggest that all cysteine cathepsins are involved in matrix degradation during endochondral ossification.