The intergenic region between the Mouse Recql4 and Lrrc14 genes functions as an evolutionary conserved bidirectional promoter.

Mammalian genomes are highly complex, with neighbouring genes arranged in divergent, convergent, tandem, antisense, and interleaving fashions. Despite the vast genomic space, a substantial portion of human genes (approximately 10%) are arranged in a divergent, head-to-head fashion and controlled by bidirectional promoters. Here we define a small core bidirectional promoter that drives expression of the mouse genes Recql4, on one strand, and Lrrc14; a novel member of the LRR gene family, on the opposite strand. Regulation of Lrrc14 expression is highly complex, involving multiple promoters' and alternative splicing. Expression of this gene is predominately restricted to neural tissue during embryogenesis and is expressed in a wide range of tissues in the adult.

Direct cell-cell interactions control apoptosis and oligodendrocyte marker expression of neuroepithelial cells.

During brain development, the neuroepithelium generates neurons and glial cells. Proliferation and differentiation of neuroepithelial cells are controlled by a complex combination of secreted factors and more intrinsic or local mechanisms, such as lateral inhibition and asymmetric division. To obtain further insights into the signals governing neuroepithelial cell fate, we used the immortomouse to derive conditionally immortalised cell lines from mouse E10 neuroepithelium. We isolated a nestin-positive basic fibroblast growth factor (bFGF)-responsive cell line (SVE10-23) which mostly differentiate into astrocytes when cocultured with primary cortical cells. We found that, by simply lowering the cell density, SVE10-23 cells embarked on oligodendrocytic differentiation as indicated by the strong expression of galactocerebroside C and 2'3'-cyclic nucleotide 3'-phosphodiesterase. Apoptosis accompanied the differentiation, and all cells died within 1 week. We present here evidence that direct interactions between cells are the main mechanism regulating this oligodendrocytic differentiation. We demonstrate that SVE10-23 cells contact or proximity inhibit their differentiation, prevent apoptosis, and promote their proliferation. Similarly, others nestin-positive precursor cell lines and nonimmortalised bFGF-grown E10 cells were found to spontaneously differentiate at low density, thus generalising the idea that neural precursor fate is regulated by direct cell-cell interactions. The SVE10-23 cell line provides a valuable tool with which to study further the molecular components implicated in this mode of regulation.

Regulation and role of Sox9 in cartilage formation.

The HMG-domain transcription factor Sox9 is a known regulator of the type II collagen gene, a major developmentally regulated protein of cartilage. In order to place Sox9 function in skeletogenesis we have investigated the regulation and misexpression of Sox9 in avian embryos. Application of exogenous BMP2 to chick limbs resulted in upregulation of Sox9, concomitant with induction of ectopic cartilage. Ectopic expression of the BMP antagonist Noggin in the limb resulted in loss of Sox9 expression from the developing digits, indicating that Sox9 expression during chondrogenesis is BMP dependent. Misexpression of Sox9 in vivo resulted in ectopic cartilage formation in limbs and in vitro was able to change the aggregation properties of limb mesenchymal cells, suggesting that Sox9 functions at the level of mesenchymal cell condensation. Misexpression of Sox9 in dermomyotomal cells, which normally give rise to the axial musculature and dermis, can result in the diversion of these cells from their normal fates towards the cartilage differentiation programme. These cells not only express type II collagen, but also Pax1, a marker of ventral fate in the developing somite. This suggests that the cell fate decision to follow the cartilage differentiation pathway is regulated at an early stage by Sox9.

Molecular cloning, chromosomal mapping, and developmental expression of a novel protein tyrosine phosphatase-like gene.

Protein tyrosine phosphatases (PTPs) mediate the dephosphorylation of phosphotyrosine. PTPs are known to be involved in many signal transduction pathways leading to cell growth, differentiation, and oncogenic transformation. We have cloned a new family of novel protein tyrosine phosphatase-like genes, the Ptpl (protein tyrosine phosphatase-like; proline instead of catalytic arginine) gene family. This gene family is composed of at least three members, and we describe here the developmental expression pattern and chromosomal location for one of these genes, Ptpla. In situ hybridization studies revealed that Ptpla expression was first detected at embryonic day 8.5 in muscle progenitors and later in differentiated muscle types: in the developing heart, throughout the liver and lungs, and in a number of neural crest derivatives including the dorsal root and trigeminal ganglia. Postnatally Ptpla was expressed in a number of adult tissues including cardiac and skeletal muscle, liver, testis, and kidney. The early expression pattern of this gene and its persistent expression in adult tissues suggest that it may have an important role in the development, differentiation, and maintenance of a number of different tissue types. The human homologue of Ptpla (PTPLA) was cloned and shown to map to 10p13-p14.

Granule cell development in the cerebellum is punctuated by changes in Sox gene expression.

Development of the vertebrate cerebellum is unusual compared to most other regions of the brain since it involves two germinal regions. Most cell types arise from the luminal, ventricular zone as in other brain regions, but granule cells arise from the second germinal layer, the external granular layer (EGL). Our analysis of the temporal and positional expression of three members of the Sox gene family of transcription factors in the cerebellum shows that granule cell development is unusual compared to most other neurons of the central nervous system (CNS). We show that granule cell precursors lose expression of cSox2 and cSox3 as they migrate to form the EGL. The EGL is the first example of a germinal layer in the CNS which does not exhibit expression of these genes. Throughout most of the CNS cSox11 expression is very low in the ventricular zone but increases dramatically as cells cease proliferation and migrate to form the subventricular zone. We also find that cSox11 expression increases when cells of the cerebellum migrate to form the EGL, but levels of expression as high as that in the subventricular zone are only seen when cells cease proliferation and migrate inwards to form the deep EGL. These observations demonstrate that cells of the proliferative superficial EGL differ qualitatively from cells of the ventricular zone in their expression of Sox genes whereas the post-proliferative cells of the deep EGL appear analogous, in their expression of Sox genes, to cells of the subventricular zone.

cSox21 exhibits a complex and dynamic pattern of transcription during embryonic development of the chick central nervous system.

cSox21 is a novel member of the Sox gene family of transcription factors. This gene is a member of the subgroup B, which includes Sox1, Sox2 and Sox3. Although all of these genes are predominantly expressed in the nervous system, only cSox21 expression is positionally restricted within the CNS. Longitudinal stripes are seen in the spinal cord and a more complex pattern is seen in the brain. The timing and position in which cSox21 stripes of expression appear provides further insight into dorsoventral patterning of the CNS. The expression of cSox21, and other genes (such as Delta, Serrate and Pax genes), may play a part in defining the developmental fate of cells along the dorsoventral axis.

Dynamic expression of chicken Sox2 and Sox3 genes in ectoderm induced to form neural tissue.

The chick genes, cSox2 and cSox3, are members of a large family of genes that encode transcription factors. Previous studies have shown that these genes are predominantly expressed in the central nervous system during embryonic development. We show that cSox3 is expressed throughout the ectoderm that is competent to form nervous tissue before neural induction. The expression of cSox3 is lost from cells as they undergo gastrulation to form nonectodermal tissues; the transcription factor, Brachyury, appears in cells about to undergo gastrulation a short time before cSox3 transcripts are lost. Therefore, Brachyury expression may act functionally upstream of cSox3 downregulation. cSox3 expression is also lost from non-neuronal ectoderm shortly after the neural plate becomes morphologically apparent. cSox2 expression increases dramatically in the central nervous system as neural ectoderm is established. The appearance of cSox2 in neural ectoderm represents one of the earliest molecular responses to neural induction documented thus far.

Embryonic expression of the chicken Sox2, Sox3 and Sox11 genes suggests an interactive role in neuronal development.

Three chicken Sox (SRY-like box) genes have been identified that show an interactive pattern of expression in the developing embryonic nervous system. cSox2 and cSox3 code for related proteins and both are predominantly expressed in the immature neural epithelium of the entire CNS of HH stage 10 to 34 embryos. cSox11 is related to cSox2 and cSox3 only by virtue of containing an SRY-like HMG-box sequence but shows extensive homology with Sox-4 at its C-terminus. cSox11 is expressed in the neural epithelium but is transiently upregulated in maturing neurons after they leave the neural epithelium. These patterns of expression suggest that Sox genes play a role in neural development and that the developmental programme from immature to mature neurons may involve switching of Sox gene expression. cSox11 also exhibits a lineage restricted pattern of expression in the peripheral nervous system.

PCR amplification of SRY-related gene sequences reveals evolutionary conservation of the SRY-box motif.

SRY (sex-determining region of the Y chromosome) has recently been identified as a key regulatory gene in mammalian sex determination. The open reading frame of this gene contains an 80-amino-acid motif, the SRY-box, which shares a high degree of homology with a DNA-binding domain found in the high-mobility-group (HMG) proteins HMG1 and HMG2. The SRY box motif is highly conserved in several sequence-specific DNA-binding proteins that are known to act as transcription factors. Here we describe the use of degenerate PCR primers to identify SRY-related sequences containing the SRY-box motif from the genomic DNA of a variety of species. The results of this study suggest that in a diverse array of species SRY-related genes may serve as transcription factors that regulate a variety of developmental pathways, including sex determination.

Distribution of endothelin-like immunoreactivity and mRNA in the developing and adult human lung.

Localization and characterization of endothelin-producing cells in the developing (fetal and postnatal) and adult human lung was investigated using the technics of immunocytochemistry and in situ hybridization. Immunoreactivity for endothelin was seen mainly in pulmonary endocrine cells of developing human lung. Immunoreactivity was also seen in the airway epithelium in fewer cases (about 50%) of human adults. In situ hybridization with 35S- or 32P-labeled RNA probes complementary to endothelin-1, -2, and -3, showed that endothelin mRNAs were expressed in a number of cells that were in similar sites to endocrine cells. Immunocytochemistry and in situ hybridization employed on pairs of reverse-face serial sections showed the presence of endothelin immunoreactivity and mRNAs in the same endocrine cell. Correlative studies revealed that endothelin is co-localized with general endocrine markers (synaptophysin, chromogranin, protein gene product 9.5) and regulatory peptides (e.g., gastrin-releasing peptide). The density (cells/mm2) of endocrine cells containing immunoreactivity or mRNAs was highest during fetal life and started to decline before birth, and was minimal in adults. Endothelin-like immunoreactivity and mRNAs were also expressed in endothelial cells. From these results, it is concluded that endothelin is synthesized in endocrine cells of human lung and the change of developmental expression of this peptide suggests it may play a part in growth regulation in addition to its putative vasoconstrictor role in human lung.