Sertoli cell differentiation and Y-chromosome activity: a developmental study of X-linked transgene activity in sex-reversed X/XSxra mouse embryos.

The requirement of Y-chromosome activity for the differentiation of somatic cells and germ cells was studied in the fetal gonads of X/XSxra mouse embryos where the activity of the Sxra fragment of the Y chromosome is influenced by the inactivation and reactivation of the X chromosome. In the interstitial somatic cells, random inactivation of the X and the XSxra chromosomes took place which was revealed by the mosaic expression of an X-linked lacZ transgene. The Sertoli cells, however, displayed a preferentially active XSxra chromosome and the presence of Sxra-active Sertoli cells was associated with the morphogenesis of testicular tubules in the sex-reversed gonads. The activity of the Y-chromosome fragment is therefore necessary for the differentiation of the Sertoli cells which may direct the development of the testis. The expression pattern of the X-linked transgene in X/XSxra germ cells suggests that both the X and the XSxra chromosomes are active. This finding suggests that the presence of Sxra has no impact on the reactivation of the X chromosome in the germ cells and that the X chromosome can be reactivated even though the germ cells are found in the testicular environment. Our results are consistent with the concept that the activity of genes on the XSxra fragment is essential for the differentiation of Sertoli cells and the morphogenesis of the testis, but not for premeiotic differentiation of germ cells in sex-reversed mice.

SOX9 directly regulates the type-II collagen gene.

Mutations in human SOX9 are associated with campomelic dysplasia (CD), characterised by skeletal malformation and XY sex reversal. During chondrogenesis in the mouse, Sox9 is co-expressed with Col2a1, the gene encoding type-II collagen, the major cartilage matrix protein. Col2a1 is therefore a candidate regulatory target of SOX9. Regulatory sequences required for chondrocyte-specific expression of the type-II collagen gene have been localized to conserved sequences in the first intron in rats, mice and humans. We show here that SOX9 protein binds specifically to sequences in the first intron of human COL2A1. Mutation of these sequences abolishes SOX9 binding and chondrocyte-specific expression of a COL2A1-driven reporter gene (COL2A1-lacZ) in transgenic mice. Furthermore, ectopic expression of Sox9 trans-activates both a COL2A1-driven reporter gene and the endogenous Col2a1 gene in transgenic mice. These results demonstrate that COL2A1 expression is directly regulated by SOX9 protein in vivo and implicate abnormal regulation of COL2A1 during, chondrogenesis as a cause of the skeletal abnormalities associated with campomelic dysplasia.

A male-specific role for SOX9 in vertebrate sex determination.

Mutation analyses of patients with campomelic dysplasia, a bone dysmorphology and XY sex reversal syndrome, indicate that the SRY-related gene SOX9 is involved in both skeletal development and sex determination. To clarify the role SOX9 plays in vertebrate sex determination, we have investigated its expression during gonad development in mouse and chicken embryos. In the mouse, high levels of Sox9 mRNA were found in male (XY) but not female (XX) genital ridges, and were localised to the sex cords of the developing testis. Purified fetal germ cells lacked Sox9 expression, indicating that Sox9 expression is specific to the Sertoli cell lineage. Sex specificity of SOX9 protein expression was confirmed using a polyclonal antiserum. The timing and cell-type specificity of Sox9 expression suggests that Sox9 may be directly regulated by SRY. Male-specific expression of cSOX9 mRNA during the sex determination period was also observed in chicken genital ridges. The conservation of sexually dimorphic expression in two vertebrate classes which have significant differences in their sex determination mechanisms, points to a fundamental role for SOX9 in testis determination in vertebrates. Sox9 expression was maintained in the mouse testis during fetal and adult life, but no expression was seen at any stage by in situ hybridisation in the developing ovary. Male-specific expression was also observed in the cells surrounding the Müllerian ducts and in the epididymis, and expression in both sexes was detected in the developing collecting ducts of the metanephric kidney. These results suggest that SOX9 may have a wider role in the development of the genitourinary system.

CD44 exhibits a cell type dependent interaction with triton X-100 insoluble, lipid rich, plasma membrane domains.

CD44 is an abundant, widely expressed transmembrane glycoprotein which can act as a receptor for the extracellular matrix glycosaminoglycan, hyaluronan. Biochemical and morphological studies have demonstrated that in fibroblasts a significant of the CD44 population is resistant to Triton X-100 extraction and that the detergent insoluble protein is co-localized with components of the cortical cytoskeleton. Surprisingly, this distribution is not abrogated upon deletion of the CD44 cytoplasmic tail indicating that mechanisms other than a direct interaction with the cytoskeleton can regulate CD44. In this manuscript, the mechanisms underlying this detergent-insoluble association are further investigated. There was no evidence that the Triton X-100 insolubility of CD44 resulted from homotypic aggregation, an association with hyaluronan or from a direct, or indirect, association with the cytoskeleton. Instead, evidence is presented that the detergent insolubility of fibroblast CD44 at 4 degrees C results from an association of the CD44 transmembrane domain with Triton X-100 resistant, lipid rich, plasma membrane domains. The proportion of the CD44 found in these Triton X-100 insoluble structures is dependent upon cell type and cannot be altered by changing cell motility or extracellular matrix associations. These studies provide evidence for a novel mechanism regulating this adhesion protein in the plasma membrane.

Induction of a hyaluronan receptor, CD44, during embryonal carcinoma and embryonic stem cell differentiation.

This paper describes the expression profile of the CD44 glycoprotein during differentiation of embryonal carcinoma (EC) and embryonic stem (ES) cells. We have recently shown that CD44 is expressed in discrete embryonic structures and, in view of this, we sought an in vitro differentiation model of development in which we could study more readily the structure and function of the CD44 molecule. The P19 EC and CGR8 ES cells were chosen as they have the capacity to develop down the cardiac muscle pathway and we have previously demonstrated that CD44 is expressed abundantly in the embryonic myocardium. The differentiation process in both cell types is accompanied by an induction of CD44 mRNA and protein. However, in differentiated cultures CD44 is not expressed in contractile cells, indicating that these P19 cells do not represent CD44-positive embryonic cardiomyocytes. Expression of CD44 is observed on fibroblast-like cells which appear to migrate over and out from the plated aggregates. Hyaluronan, the major ligand for CD44, is also associated with these CD44-positive fibroblast-like cells. It is suggested that expression of both receptor and ligand by the fibroblast cells is required for cell:matrix adhesion and cell motility. As CD44 is up-regulated in these cultures, P19 cells are now established as a useful model system to study the factors regulating expression of the CD44 gene.

Restricted expression of the hyaluronan receptor, CD44, during postimplantation mouse embryogenesis suggests key roles in tissue formation and patterning.

CD44 is a multifunctional adhesion protein that acts as a major receptor for the hygroscopic extracellular matrix component, hyaluronan. This receptor-ligand binding directly mediates at least some of the cell-cell and cell-matrix interactions ascribed to CD44. Other interactions involving CD44 may be modulated indirectly by its ability to bind growth factors and thereby to promote cell attachment. During vertebrate development, multiple cases of hyaluronan involvement in cell proliferation, cell migration and histogenesis have been documented. In addition, there is evidence suggesting a central role for cell surface glycoproteins and proteoglycans in mediating the action of polypeptide growth factors involved in tissue patterning. In view of this, we undertook to investigate expression of the CD44 protein during postimplantation mouse embryogenesis. Between 9.5 and 12.5 days of embryonic development, the predominant form of CD44 protein corresponds to the hyaluronan-binding CD44H form. However, species with a higher M(r) were also detected, implying that CD44 isoforms generated by alternative splicing of CD44 RNA are employed in normal development. Further, we used mouse embryos to perform whole-mount immunohistochemistry and examine the temporal and spatial distribution of this glycoprotein. CD44 is expressed at high levels in the heart, somites and condensing limb-bud mesenchyme at critical stages of morphogenesis. These sites correlate with regions where hyaluronan has been demonstrated to regulate morphogenetic events. Of novel interest, however, is the high expression of CD44 in regions that do not correlate with sites of known hyaluronan-mediated developmental events. These include instructive epithelia participating in epithelial-mesenchymal cell interactions such as the apical ectodermal ridge of the developing limb bud and the odontogenic placodes of the presumptive upper and lower jaws.

Redistribution of secretory granule components precedes that of synaptic vesicle proteins during differentiation of a neuronal cell line in serum-free medium.

Incubation of the rat sensory neuron-derived cell line ND7 in serum-free medium results in the arrest of mitosis and the appearance of numerous neuronal processes. During this differentiation event, secretory granule components such as chromogranins, neuropeptide Y and the C-flanking peptide of pro-neuropeptide Y move to the tips of the majority of the neuronal processes regardless of process length. In contrast, the synaptic vesicle component, synaptophysin, is found only at the tips of the very long processes which appear following prolonged periods of culture in serum-free medium. A similar restriction of synaptophysin to long processes is also observed following differentiation and process formation induced by other treatments such as incubation in reduced serum or treatment with cyclic AMP or phorbol myristate acetate. Hence the regulated secretory pathway associated with the chromogranins and neuropeptides appears to be segregated into the processes at an earlier stage of ND7 differentiation than the synaptophysin-associated synaptic vesicle pathway. ND7 cells therefore provide a model system for studying the processes regulating these pathways and the redistribution of their components during neuronal differentiation.

Elevation of cyclic AMP levels in cell lines derived from latently infectable sensory neurons increases their permissivity for herpes virus infection by activating the viral immediate-early 1 gene promoter.

Immortalized cell lines derived from sensory neurons are relatively non-permissive for lytic infection with herpes simplex virus (HSV) and fail to transcribe the viral immediate-early genes following infection. Treatment of these cells with agents which raise the intra-cellular level of cyclic AMP results in increased activity of the IE1 gene which contains a cyclic AMP response element within its promoter and produces a consequent increase in permissivity for HSV infection. The significance of these effects for the regulation of HSV infection of neuronal cells are discussed in the light of the finding that cyclic AMP treatment can reactivate latent HSV infections.

Intracellular redistribution of neuropeptides and secretory proteins during differentiation of neuronal cell lines.

We have demonstrated that the mouse neuroblastoma N18Tg2 cell line and several clones of hybrid ND cells (ND7, ND9 and ND21), derived from the fusion of neonatal rat sensory neurons with that neuroblastoma, show immunostaining to protein gene product 9.5, neuropeptide Y, C-flanking peptide of neuropeptide Y, tyrosine hydroxylase and chromogranins. Synaptophysin could only be detected in ND cells. Immunoreactivities to substance P, calcitonin gene-related peptide, galanin and somatostatin could not be detected in any of these cell lines. After three days of incubation in a differentiation medium, cell processes of various lengths were observed both in neuroblastoma and ND cell cultures. In ND7 cells there was also a redistribution of neuropeptide Y and its C-flanking peptide to the tips of cell processes. The differentiation of cell processes was also accompanied by the appearance of immunostaining to rat chromogranins in their tips. In contrast, synaptophysin expression was found mainly in cell bodies. Neuropeptide Y, its C-flanking peptide and chromogranins have been associated with secretory granules, whereas synaptophysin is a marker for small synaptic-like vesicles. Therefore, our morphological findings further support and expand the view that these markers are primarily associated with different subcellular structures. Moreover, they indicate that the regulated secretory pathway associated with chromogranins is segregated into nerve processes at an early stage of differentiation, when the synaptophysin-associated pathway is not yet mature. ND7 cells thus provide a useful model system for studying changes in the distribution of neuropeptides, cytoskeletal elements and proteins associated with cell secretion during neuronal differentiation.

The octamer-binding protein Oct-2 represses HSV immediate-early genes in cell lines derived from latently infectable sensory neurons.

Transcription of herpes simplex virus (HSV) immediate-early (IE) genes does not occur in sensory neurons latently infected with the virus or following infection of neuronal cell lines. In neuronal cell lines this inability results from the weak activity of the viral IE promoters, which is caused by a neuron-specific repressor factor that binds specifically to the TAATGARAT motif in these promoters and to related octamer elements. Cells expressing this repressor contain an additional octamer-binding protein that is absent from permissive cells. We identify this factor as the lymphocyte- and neuron-specific octamer-binding protein Oct-2 and show that Oct-2 mRNA is also present in dorsal root ganglion neurons, the natural site of HSV latency in vivo. Moreover, artificially elevated expression of Oct-2 can repress the IE promoter. The potential role of Oct-2 in the initiation and maintenance of in vivo latent infection with HSV is discussed.

A cellular factor binding to the TAATGARAT DNA sequence prevents the expression of the HSV immediate-early genes following infection of nonpermissive cell lines derived from dorsal root ganglion neurons.

Cell lines derived from dorsal root ganglion neurons are nonpermissive for HSV infection and do not transcribe the viral immediate-early genes following infection. The lack of immediate-early gene transcription in these cells is caused by the presence of a neuronal cell specific inhibitory factor which binds to the TAATGARAT elements in the promoters of these genes and prevents their transcription. The significance of these results for an understanding of the processes regulating the interaction of HSV with neuronal cell types and the establishment of latent infections in vivo is discussed.

Cell lines derived from dorsal root ganglion neurons are nonpermissive for HSV and express only the latency-associated transcript following infection.

The effect of herpes simplex virus type 1 infection in a series of immortalized dorsal root ganglion cell lines has been investigated. Following infection of one of these lines, the viral immediate-early genes are not transcribed and the lytic cycle is aborted at an early stage. In contrast these cells do support transcription of the gene encoding the latency-associated transcripts which are the only viral RNAs present in latently infected ganglia in vivo. These cell lines are therefore a suitable model system for studies of the processes regulating the interaction of HSV with neuronal cell types and the establishment of latent infections in vivo.

Activity of herpes simplex virus type 1 latency-associated transcript (LAT) promoter in neuron-derived cells: evidence for neuron specificity and for a large LAT transcript.

By using chloramphenicol acetyltransferase (CAT) assays in neuron-derived cell lines, we show here that promoter activity associated with the herpes simplex virus type 1 latency-associated transcript (LAT) had neuronal specificity. Promoter activity in these transient CAT assays coincided with a DNA region containing excellent RNA polymerase II promoter consensus sequences. Primer extension analysis in a LAT promoter-CAT plasmid construct placed the start of transcription about 28 nucleotides from the first T in the consensus TATA box sequence. Neuronal specificity of this promoter was suggested by examining the effect of sequences upstream of the promoter on CAT activity in neuronal versus nonneuronal cells. In nonneuronal cells, promoter activity was decreased 3- to 12-fold with the addition of upstream sequences. In contrast, in neuron-derived cells, the addition of upstream sequences did not decrease promoter activity. The LAT promoter predicted by our transient CAT assays was located over 660 nucleotides upstream from the 5' end of the previously mapped 2-kilobase (kb) LAT. This unusual location was explained by in situ and Northern (RNA) blot hybridization analyses that suggested that LAT transcription began near the promoter detected in our CAT assays, rather than near the 5' end of the 2-kb LAT. In situ hybridization with neurons from latently infected rabbits detected small amounts of LAT RNA within 30 nucleotides of the consensus TATA box sequence. This suggested that LAT transcription began near this TATA box. Northern blot hybridization of RNA from ganglia of latently infected rabbits revealed a faint 8.3-kb band of the same sense as LAT. We conclude that (i) the LAT promoter has neuronal specificity, (ii) the LAT promoter is located over 660 nucleotides upstream of the 5' end of the previously characterized stable 2-kb LAT, (iii) LAT transcription begins about 28 nucleotides from the first T of the consensus TATA box sequence and extends to near the first available polyadenylation site approximately 8.3 kb away, and (iv) this 8.3-kb RNA may be an unstable precursor of the more stable 2- and 1.3-kb LATs.