Cell Type-specific Expression Patterns Research Articles

HLA‐G Transgenic Mice: A Model for Studying Expression and Function at the Maternal/Fetal Interface

Experiments designed to identify all HLA class I genes led to the cloning of the HLA-G gene (Geraghty et al. 1987). Very low levels of HLA-G mRNA expression have been demonstrated in the eye, thymus, peripheral blood lymphocytes and in keratinocytes (Shukla et al. 1990, Ishitani & Geraghty 1992, Kirszenbaum et al. 1994, Ulbrecht et al. 1994). Higher levels of HLA-G expression were demonstrated in certain subpopulations of trophoblasts (Ellis et al. 1990, Kovats et al. 1990). Specifically, those subpopulations of trophoblasts in direct contact with decidua, i.e. the cytotrophoblasts of the cytotrophoblast shell and columns, invasive cytotrophoblasts, and cytotrophoblasts of the chorionic membrane, express HLA-G (Yelavarthi et al. 1991, Chumbley et al. 1993). HLA-G molecules on these cells are in a position to interact directly with the maternal immune system. HLA-G transgenic mice have been produced in an effort to produce a model in which antigen presentation by HLA-G can be studied. Studies utilizing these transgenic mice have led to the definition of a previously unknown regulatory region and have yielded some basic information about HLA-G's ability to function as a class I molecule (Schmidt et al., in press; manuscript in preparation). One of the HLA-G transgenic mouse lines produced, G.3.2, has been shown to have a cell-type specific extraembryonic HLA-G expression pattern paralleling that seen in human extraembryonic tissues (Schmidt et al, in press). In addition, the levels of HLA-G mRNA expression seen in the extraembryonic tissues from this transgenic mouse line are similar to the levels of HLA-G mRNA expression detectable in human extraembryonic tissues. As cells expressing HLA-G come in direct contact with decidual cells, these mice will serve as a model in which HLA-G's ability to present foreign antigens to the maternal immune system can be studied. The G.3.2 HLA-G transgenic mouse line also exhibits thymic HLA-G expression levels similar to those seen in human thymus (Schmidt et al., manuscript in preparation). HLA-G expression in the transgenic murine thymus is largely restricted to dendritic cells. It is possible that this thymic HLA-G mRNA expression is responsible for the tolerance to HLA-G which is seen in the HLA-G transgenic mice. Further studies will be necessary to determine whether the lymphocytic repertoire of the HLA-G transgenic mouse can recognize HLA-G molecules presenting foreign peptides.

Overlapping and differential expression of BIG‐2, BIG‐1, TAG‐1, and F3: Four members of an axon‐associated cell adhesion molecule subgroup of the immunoglobulin superfamily

Axon-associated cell adhesion molecules (AxCAMs) play crucial roles in the formation, maintenance, and plasticity of functional neuronal networks. We report here a molecular cloning of a novel AxCAM, BIG-2. BIG-2 is a member of TAG-1/F3 subgroup of the immunoglobulin (Ig) superfamily, with six Ig-like domains, four fibronectin type III-like repeats, and a glycosyl phosphatidylinositol-anchoring domain. Recombinant BIG-2 protein had a neurite outgrowth-promoting activity when used as a substrate for neurons in vitro. To survey the spatial expression pattern of BIG-2 in comparison with other TAG-1/F3 subgroup members, an in situ hybridization analysis was performed in adult and developing rat brain sections with riboprobes specific for BIG-2, BIG-1, TAG-1, and F3. The four AxCAM transcripts displayed cell type-specific expression patterns with overlapping and distinct profiles. In adult hippocampus, for example, we observed BIG-1 mRNA specifically in granule cells of the dentate gyrus, BIG-2 mRNA highly in the CA1 pyramidal cells, TAG-1 mRNA predominantly in the CA3 pyramidal cells, and F3 mRNA in neurons in all of these fields. These results suggest that BIG-2, BIG-1, TAG-1, and F3 may play important roles in the formation and maintenance of specific neuronal networks in the brain.

Expression of Notch 1, 2 and 3 is regulated by epithelial-mesenchymal interactions and retinoic acid in the developing mouse tooth and associated with determination of ameloblast cell fate.

Notch 1, Notch 2, and Notch 3 are three highly conserved mammalian homologues of the Drosophila Notch gene, which encodes a transmembrane protein important for various cell fate decisions during development. Little is yet known about regulation of mammalian Notch gene expression, and this issue has been addressed in the developing rodent tooth during normal morphogenesis and after experimental manipulation. Notch 1, 2, and 3 genes show distinct cell-type specific expression patterns. Most notably, Notch expression is absent in epithelial cells in close contact with mesenchyme, which may be important for acquisition of the ameloblast fate. This reveals a previously unknown prepatterning of dental epithelium at early stages, and suggests that mesenchyme negatively regulates Notch expression in epithelium. This hypothesis has been tested in homo- and heterotypic explant experiments in vitro. The data show that Notch expression is downregulated in dental epithelial cells juxtaposed to mesenchyme, indicating that dental epithelium needs a mesenchyme-derived signal in order to maintain the downregulation of Notch. Finally, Notch expression in dental mesenchyme is upregulated in a region surrounding beads soaked in retinoic acid (50-100 micrograms/ml) but not in fibroblast growth factor-2 (100-250 micrograms/ml). The response to retinoic acid was seen in explants of 11-12-d old mouse embryos but not in older embryos. These data suggest that Notch genes may be involved in mediating some of the biological effects of retinoic acid during normal development and after teratogenic exposure.

Cell Type-specific Transactivation of the VCAM-1 Promoter through an NF-κB Enhancer Motif

Cytokine activation of vascular cell adhesion molecule-1 (VCAM-1) gene expression by endothelial cells is an important feature in a variety of vascular inflammatory responses. Cytokines transcriptionally activate the VCAM-1 promoter in endothelial cells at least in part through two closely linked NF-kappa B enhancer motifs, kappa L-kappa R (positions -77 and -63). However, cytokine activation of the dimeric NF-kappa B transcriptional factor (p50+p65 subunits) occurs in almost all cell types, whereas VCAM-1 gene expression exhibits a cell type-specific pattern of expression. Tumor necrosis factor-alpha markedly transactivated a transiently transfected minimal kappa L-kappa R motif-driven VCAM-1 promoter, p85VCAMCAT, in passaged human vascular endothelial cells but not in the human epithelial cell line, HeLa suggesting that cell type-specific factors may function through the kappa L-kappa R motif. Both cell types exhibited similar inductions of NF-kappa DNA binding activity and transcriptional activity. However, co-transfection of HeLa cells with p65 and p50 expression vectors demonstrated that the minimal VCAM-1 promoter was effectively transactivated by p65 alone but that additional co-expression of p50 blocked this activity. Furthermore, cytokine activation of the minimal VCAM-1 promoter in HeLa cells was recovered by inhibition of p50 expression using antisense oligonucleotide. These studies suggest that the NF-kappa B(p50+p65 heterodimer) does not support transactivation of the VCAM-1 promoter with the p50 subunit potentially playing a significant inhibitory role in suppressing cytokine activation of VCAM-1. In addition, p65 associated transcriptional factors other than NF-kappa B may serve as positive, cytokine-inducible, cell type-specific regulators of VCAM-1 gene expression.

Fibroblast growth factors in the nervous system.

Fibroblast growth factors (FGFs) exhibit widespread mitogenic and neurotrophic activities. Nine members of the family are currently known, and FGF-1 and FGF-2 are present in relatively high levels in CNS. FGF-1 is expressed by a subset of neuronal populations, while FGF-2 is expressed by astrocytes. FGF-1 and FGF-2 lack signal peptides and appear to be present mainly in intracellular compartments. This suggests that the factors may act as initiators of a repair response after injury. Support for this notion comes from observations that FGF-1 and FGF-2 levels are low during critical phases of development, but high in the adult CNS. A family of transmembrane tyrosine kinase receptors (FGFRs) mediates the effects of FGFs. Four different genes coding for FGF receptors are currently known, three of which are expressed in cell type-specific patterns in the CNS. The main receptor variants present in this tissue, however, can by themselves not distinguish between FGF-1 and FGF-2. Additional selectivity may be established by interaction of the FGFs and their receptors with select heparan proteoglycans (HSPGs). Therefore, the precise physiological role of FGFs is determined by the combination of cell type-specific patterns of expression of FGFs, FGFRs and HSPGs together with the mechanisms that regulate the extracellular availability of FGFs.

The rabbit gene for 92-kDa matrix metalloproteinase. Role of AP1 and AP2 in cell type-specific transcription.

We isolated the rabbit gene for the 92-kDa matrix metalloproteinase, gelatinase B, and sequenced 1802 contiguous bases covering the first three exons and 522 bases of DNA upstream of the start site for transcription. The DNA between bases -519 and +19 is sufficient to drive expression of a reporter gene in early passage cultures of corneal fibroblasts or primary cultures of corneal epithelial cells. Basal activity of the gelatinase B promoter in fibroblasts is lower than a collagenase promotor of 1800 base pairs, but activity of both promotors is similarly stimulated by treatment of transfected cells with phorbol 12-myristate 13-acetate, and stimulation is enhanced by co-treatment with transforming growth factor-beta. In contrast, basal activity of the gelatinase B promotor in epithelial cells is higher than the collagenase promotor. Deletion analysis demonstrated that sequences upstream of base -330 confer cell type-specific activity to the gelatinase B promotor. Site-directed mutagenesis revealed that an AP1-like element within this region is specifically utilized by fibroblasts. This region also contains elements that confer the capacity for activation by AP2, a transcription factor found to be expressed by corneal epithelial cells but not by corneal fibroblasts. In contrast, AP2 does not activate the collagenase promotor. These results provide a molecular basis for the unique cell type-specific expression pattern of gelatinase B as compared to other matrix metalloproteinases.

Sequences Distal to the AP1/E Box Motif Are Involved in the Cell Type‐Specific Expression of the Rat Tyrosine Hydroxylase Gene

In order to define cell type-specific elements associated with the catecholamine biosynthetic enzyme, tyrosine hydroxylase (TH), transient transfections of promoter deletion constructs were used to test relative reporter-gene activities in TH-expressing and -nonexpressing cell lines. Such assays demonstrated that a region between -503 and -578 contributed to rat TH promoter activity in the pheochromocytoma cell line PC12. Deletion of these sequences resulted in a 66% loss in cell type-specific activity. Mutations within the E box/dyad symmetry element (CAGGTGCCTGTGACAGTG) did not affect the basal and cell type-specific pattern of expression exhibited by the rat TH promoter. Promoter fusion constructs between the rat TH promoter (-741 and -197) and the human TH promoter (-197 and +1) exhibited reporter-gene activities equivalent to that of wild-type -741 rat TH constructs, further demonstrating that sequence elements upstream of the rat E box/dyad symmetry are important for cell type-specific expression. Gel-shift experiments indicated that a PC12 nuclear factor could bind to a 39-bp sequence within this region in a cell type-specific manner. The size of this factor was 52 kDa as determined by UV cross-linking experiments.

Ectopic expression of beta-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation and in situ localization.

We produced transgenic mice carrying the native sheep beta-lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains. In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.

Differential induction of Pax genes by NGF and BDNF in cerebellar primary cultures.

The Pax genes encode sequence-specific DNA binding transcription factors that are expressed in embryonic development of the nervous system. Primary neuronal cell cultures derived from the cerebellar cortex of embryonic day 14, newborn and 7-d old mice, were used to investigate the cell-type specific expression patterns of three members of the murine paired box containing gene family (Pax gene family), in vitro. Cell types which express Pax-2, Pax-3, and Pax-6 RNA in primary cultures correspond to those found in regions of the cerebellum which show RNA signals in sections of the developing mouse brain. To find mechanisms regulating Pax gene expression during cerebellar development, the differential regulation of Pax-2, Pax-3, and Pax-6 by NGF and BDNF, two structurally related neurotrophins, was studied in such primary cultures. Pax-2 and Pax-6 RNA increased slightly by 1 h and remained elevated throughout a 24-h treatment with BDNF and NGF. Pax-3 RNA was not detected in newborn cultures, but underwent a rapid (1 h) and transient (2 h) induction upon treatment with either BDNF or NGF. No response was seen with EGF or FGF. Cycloheximide treatment amplified Pax-3 induction and prolonged the signal. Thus, Pax-3 induction resembles that of the immediate-early gene c-fos, which transduces growth factor signals during the development of particular neuronal/glial cell types. The changes in Pax expression were inductive rather than trophic.

Presence of a nuclear lamina in pachytene spermatocytes of the rat

The nuclear lamina is a karyoskeletal structure located at the periphery of cell nuclei. The major constituents are the lamins, which belong to the evolutionarily conserved multigene family of intermediate filament proteins. Lamins show a conspicuous cell type-specific expression pattern. The majority of somatic cells of vertebrates express A-type (lamins A and C) as well as B-type (lamins B1 and B2) lamins. Although a lamina structure has been demonstrated to be a ubiquitous component of somatic nuclei its existence in certain meiotic stages during spermatogenesis has been a matter of debate. In this study, we investigated the expression of lamins in rat spermatogenic cells using immunological and protein-chemical methods. We report on the presence of a nuclear lamina structure in rat pachytene spermatocytes. With the aid of a novel broad-reacting lamin antibody we have demonstrated the expression of a protein that is closely related, if not identical, to lamin B1.

The ubiquitous transactivator Zfp-38 is upregulated during spermatogenesis with differential transcription

We describe the complete nucleotide sequence of a full length cDNA clone encoding a new mouse zinc finger protein gene, Zfp-38 and localize it on chromosome 5 by the interspecific backcross analysis. The N-terminal domain of the Zfp-38 protein (64 kDa) contains 358 amino acids and the C-terminal domain of 197 residues encodes 7 zinc fingers. We also present evidence that Zfp-38 is a strong transcriptional activator. The transactivation domain was localized in the non finger region and a fusion protein containing 112 amino acid residues from this region of the Zfp-38 and the DNA binding domain of the yeast Gal 4 protein, very efficiently transactivated the expression of a reported CAT plasmid, harboring the Gal4 target site. By in situ hybridization and northern blotting technique, the Zfp-38 transcript can be detected at a highly elevated level during spermatogenesis. Its expression accompanies the progression from pachytene spermatocytes to round spermatids. The undifferentiated spermatogonia or the haploid elongated spermatid and the spermatozoa do not show detectable level of the transcript. Interestingly, other tissues express low levels of a slightly shorter transcript with a different 5′ end as determined by RNase protection. The presence of both a transcriptional activating domain and 7 DNA binding zinc fingers, coupled with the cell type(s) specific expression pattern, suggests that Zfp-38 has the potential to regulate transcription during spermatogenesis.

Identification of a short nuclear lamin protein selectively expressed during meiotic stages of rat spermatogenesis

Abstract The nuclear lamina is a karyoskeletal structure located at the nuclear periphery and intimately associated with the inner nuclear membrane. It is composed of a multigene family of proteins, the lamins, which show a conspicuous cell type-specific expression pattern. The functional role of lamins has not been definitively established but available information indicates that they are involved in the organization of nuclear envelope and interphase chromatin. Spermatogenesis is characterized, among other features, by stage-specific changes in chromatin organization and function. These changes are accompanied by modifications in the organization and composition of the nuclear lamina. In previous experiments we have determined that rat spermatogenic cells express a lamin closely related, if not identical, to lamin B1 of somatic cells; whereas rat somatic lamins A, C, D and E were not detected. Considering that chromatin reorganizations during spermatogenesis may be directly or indirectly related to changes of the nuclear lamina we have decided to further investigate lamin expression during this process. Here we report on the identification of a 52 kDa protein of the rat which, according to immunocytochemical and biochemical data, appears to be a novel nuclear lamin. Using meiotic stage-specific markers, we have also demonstrated that this short lamin is selectively expressed during meiotic stages of spermatogenesis.

Tenascin in bone morphogenesis: expression by osteoblasts and cell type-specific expression of splice variants.

The extracellular matrix glycoprotein, tenascin, is associated in vivo with mesenchyme undergoing osteogenesis and chondrogenesis, but is absent from mature bone and cartilage matrix. The expression of tenascin by osteoblastic cells in vitro has been investigated by immunoblotting and immunocytochemistry. Tenascin was secreted into the medium and deposited in the matrix by human and rat osteoblast-like cell lines, as well as by primary osteoblast-enriched cultures from chick embryo calvarial bones. In primary osteoblast-enriched cultures, extracellular tenascin was found only in cell aggregates expressing the osteoblast marker alkaline phosphatase. Chicken osteoblast cultures synthesized almost exclusively the largest tenascin subunit, whereas fibroblast cultures from periostea of chicken calvariae synthesized approximately equal amounts of all three subunits. In situ hybridization studies of developing chicken bones, using a cDNA probe that hybridizes to all chicken tenascin splice variants, showed specific labelling of both osteogenic and chondrogenic regions of developing endochondral bones. In contrast, a cDNA probe specific for the large tenascin splice variant showed specific hybridization in osteogenic but not chondrogenic regions. Within osteogenic regions, tenascin mRNA was expressed by osteoblasts. A comparison of in situ hybridization and immunohistochemical studies demonstrated that tenascin mRNA and protein were codistributed in osteogenic regions of endochondral and membrane bones, whereas protein was retained in regions of differentiating cartilage where mRNA was no longer detectable. The results presented here demonstrate that tenascin is synthesized by osteoblasts. Moreover, within developing bones, there are at least three different cell type-specific patterns of expression of tenascin splice variants.

The human IGF-I gene contains two cell type-specifically regulated promoters

The human insulin-like growth factor-I (IGF-I) gene contains two alternative leader exons: exons 1 and 2. We have identified, by transient transfection experiments, the putative promoters P1 and P2 upstream of these leader exons. The promoter regions were cloned in front of the luciferase reporter gene and their promoter activities were measured in transfected SK-N-MC (human neuroepithelioma) and OVCAR-3 (human ovarian carcinoma) cells. Both of these cell lines express the IGF-I gene endogenously, resulting in normally sized IGF-I mRNAs of 7.6, 1.3 and 1.1 kb. In SK-N-MC cells, in which P1 is the most active IGF-I promoter, P2 displayed a three times lower promoter activity than P1. However, in OVCAR-3 cells, P2 is four times more active than P1, resulting in an overall 12-fold difference in the relative promoter activities of the two IGF-I gene promoters in these two cell types. This indicates that the IGF-I promoters show a cell type-specific expression pattern.

LEF-1, a gene encoding a lymphoid-specific protein with an HMG domain, regulates T-cell receptor alpha enhancer function [corrected

Lymphoid-specific cDNA clones were isolated that encode a nuclear protein with homology to the chromosomal nonhistone protein HMG-1 and to putative regulators of cell specialization, including the mammalian testis-determining factor SRY and fungal mating-type proteins. The gene represented by the isolated cDNA clones, termed LEF-1 (lymphoid enhancer-binding factor 1), is developmentally regulated and expressed in pre-B and T lymphocytes but not in later-stage B cells or nonlymphoid tissues. Both endogenous and recombinant LEF-1 were shown to bind to a functionally important site in the T-cell antigen receptor (TCR) alpha enhancer. Maximal TCR alpha enhancer activity was found to parallel the cell type-specific expression pattern of LEF-1. Moreover, forced expression of recombinant LEF-1 in late stage B cells increases TCR alpha enhancer function. Taken together, these data suggest that LEF-1 is a regulatory participant in lymphocyte gene expression and differentiation.