RESUMEN
Neural crest cells migrate along specific pathways to their destinations and, like neuronal growth cones, must be guided by extracellular cues. One example of neural crest pathfinding is the segmental migration of branchial and trunk neural crest; this is associated with the patterning of the skeletal components of the branchial arches and of the peripheral nervous system. In this review, we discuss recent work that has implicated Eph receptors and their ephrin ligands in mediating repulsive interactions that restrict neural crest cell migration. We relate these findings to the roles of these receptors and ligands in growth cone guidance and the segmental restriction of cell movement in the hindbrain.
Asunto(s)
Movimiento Celular/fisiología , Cresta Neural/citología , Cresta Neural/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Animales , Humanos , Ligandos , Neuronas/citología , Neuronas/fisiologíaRESUMEN
Recent studies have implicated Eph-related receptor tyrosine kinases and their membrane-bound ligands in restricting or stimulating the movement of cells and axons. Members of these large families of receptors and ligands fall into two major binding specificity classes, in which the GPI-anchored subgroup of ligands can each bind to all members of a subgroup of receptors, whereas the transmembrane ligands interact with a distinct subgroup of receptors. Analysis of expression patterns is therefore important in order to understand which receptor-ligand interactions occur in vivo. We have cloned mouse orthologues of five members of the ligand family and analysed in detail their developmental expression, in comparison with each other, and with the receptor specificity class they can interact with. We find that B61, AL-1/RAGS, LERK4, and ELF-1, members of the GPI-anchored subgroup of ligands, have both distinct and overlapping aspects to their expression in early mesoderm, somites, and branchial arches; in complex, dynamic patterns in the limb; and in spatial domains and specific neurons in the CNS. Similarly, Elk-L is expressed in hindbrain segments, the roof plate, and floor plate, which overlaps with that of other transmembrane ligands, but has distinct expression in somites. The expression domains of ligands are complementary to those of the corresponding receptors in a number of tissues, including the midbrain, hindbrain, and differentiating limbs, consistent with potential roles in restricting cell movement. In addition, we find that there are some overlaps in expression of receptors and ligands, for example in somites and the early limb. Taken together with previous studies showing that Eph-related receptors also have distinct but overlapping expression patterns, these data indicate that each ligand may have stage- and tissue-specific interactions with an individual member or multiple members of the receptor family.
Asunto(s)
Desarrollo Embrionario y Fetal , Regulación del Desarrollo de la Expresión Génica , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Secuencia de Aminoácidos , Animales , Ligandos , Proteínas de la Membrana/biosíntesis , Ratones , Datos de Secuencia Molecular , Proteínas Tirosina Quinasas Receptoras/genética , Receptor EphB6RESUMEN
WT1 is a Wilms' tumor suppressor gene that maps to human chromosome 11p13 and encodes a putative transcription factor implicated in controlling normal urogenital development. Sporadic homozygous mutations in WT1 result in the development of Wilms' tumor (nephroblastoma), and heterozygous germline mutations can give rise to a phenotype which includes nephropathy and urogenital abnormalities (the Denys-Drash syndrome). Thus, inappropriate expression of WT1 results in developmental abnormalities affecting the urogenital system. To better define the temporal and spatial distribution of WT1 expression during embryogenesis, we have used in situ mRNA hybridization and immunohistochemistry to examine WT1 expression in murine embryos during the period prior to and throughout active organogenesis. Prior to embryological day 9.5 (E9.5), WT1 mRNA expression is absent in the embryo proper but is strongly expressed in the maternal uterus. During the initiation of organogenesis on E10.5, WT1 mRNA is localized within the pronephric and mesonephric tissues. By E11.5, the nephrogenic cord, urogenital ridge, and condensing metanephric tissue show intense WT1 hybridization signals, and increasingly centripetal expression of WT1 in the kidney correlates with renal differentiation from days E11.5 through E16.5. The stromal cell components in the developing gonad show expression of WT1 by E10.5, whereas in the remaining organs examined, WT1 expression is restricted to the uterus, spleen, abdominal wall musculature, and mesothelial lining of organs within the thoracic and abdominal cavities. Interestingly, there is also WT1 expression in the central nervous system which localizes to the ependymal layer of the ventral aspect of the spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Desarrollo Embrionario y Fetal/genética , Genes del Tumor de Wilms , Transcripción Genética , Animales , Técnica del Anticuerpo Fluorescente , Gástrula/metabolismo , Expresión Génica , Hibridación in Situ , Ratones , Sistema Nervioso/embriologíaRESUMEN
We have identified elements in the 5' region of the murine tissue inhibitor of metalloproteinase (TIMP) gene which control the response to serum, phorbol esters and transforming growth factor beta (TGF-beta) in cell culture. These elements lie between -858 and -601 relative to the translation initiation start site in the gene and are distinct from those that control expression in response to virus induction. The temporal and spatial expression of the TIMP gene was also analysed during mouse embryogenesis using in situ hybridization. A transgenic mouse line was constructed which expressed a TIMP/lac Z fusion gene. Using in situ beta-galactosidase assays we were able to compare the expression of the transgene with the endogenous gene. Thus we concluded that most of the sequences controlling in vivo expression of TIMP were present in the transgene and could be localised to the 5' region of the gene.
Asunto(s)
Desarrollo Embrionario y Fetal/genética , Regulación de la Expresión Génica , Glicoproteínas/biosíntesis , Células 3T3 , Animales , Secuencia de Bases , Células Cultivadas , Matriz Extracelular/metabolismo , Genes Sintéticos , Glicoproteínas/genética , Humanos , Hibridación in Situ , Ratones , Ratones Transgénicos/embriología , Ratones Transgénicos/genética , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Inhibidores Tisulares de MetaloproteinasasRESUMEN
Tissue inhibitor of metalloproteinase (TIMP) is one of a family of metalloproteinase inhibitors and a major interstitial inhibitor of collagenase. Transcription of the TIMP gene is induced by such diverse agents as viruses, phorbol esters, serum, and growth factors. We have previously assigned the regulatory elements responsible for induction of transcription in response to viruses to the first intron of the murine TIMP gene. Here we have identified a promoter and an enhancer element responsive to serum and the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. Based on a comparative sequence analysis of the murine and human genes, the enhancer element is part of a 38-base pair conserved sequence. Gel mobility shift assays indicate that this enhancer is a phorbol ester-responsive-like element that likely binds one of a family of AP-1 proteins. Interestingly, the region containing the phorbol ester-responsive-like element is also sufficient to direct a response to transforming growth factor beta 1 in the presence of serum.
Asunto(s)
Glicoproteínas/genética , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Secuencia de Bases , Sangre , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Deleción Cromosómica , Medios de Cultivo , Elementos de Facilitación Genéticos , Humanos , Intrones , Ratones , Colagenasa Microbiana/antagonistas & inhibidores , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , ARN Mensajero/genética , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Inhibidores Tisulares de Metaloproteinasas , Transfección , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
TIMP (tissue inhibitor of metalloproteinase) is a glycoprotein inhibitor of metalloproteinases that we hypothesize to be involved in the tissue remodeling that occurs during each hair growth cycle. We examined this hypothesis by studying the expression of TIMP at selected times during a single hair cycle using TIMP-lacZ transgenic mice to localize TIMP gene activity in the hair follicle. TIMP gene induction was visualized by staining mouse back skin for beta-galactosidase (beta-gal) activity. Paraffin sections were analyzed for the localization of TIMP expression. TIMP gene activation appears in hair follicles only during the mid-anagen (the growing stage of the hair cycle) primarily in Henle's layer of the inner root sheath. Some expression of TIMP is also seen in a few connective tissue cells, in the sebaceous gland and in cells at the proximity of the dermal papilla cells in catagen (regressing) and telogen (resting) follicles. These results are consistent with a role for TIMP in cyclic remodeling of connective tissue in hair follicles.
Asunto(s)
Glicoproteínas/análisis , Cabello/química , Metaloendopeptidasas/antagonistas & inhibidores , Animales , Tejido Conectivo/fisiología , Glicoproteínas/fisiología , Cabello/crecimiento & desarrollo , Ratones , Ratones Transgénicos , Inhibidores Tisulares de Metaloproteinasas , beta-Galactosidasa/análisisRESUMEN
X chromosome inactivation results in the cis-limited inactivation of most, but not all, genes on one of the two X chromosomes in mammalian females. The molecular basis for inactivation is unknown. In order to examine the transcriptional activity of human X-linked genes, a series of mouse-human somatic cell hybrids under positive selection for the active or inactive human X chromosome has been created. Northern blot analysis of RNA from these hybrids showed that the human MIC2 gene, which is known to escape X inactivation, was transcribed in hybrids with either the active or inactive X chromosome. In contrast, the human TIMP gene was only transcribed in hybrids with an active human X chromosome. Further analysis using the polymerase chain reaction showed that there was at least one-hundred fold less transcription of the TIMP gene from the inactive X than from the active X chromosome. These findings demonstrate that the human TIMP gene is subject to X inactivation at the level of transcription, and illustrate the usefulness of the polymerase chain reaction to study the extent of X-linked gene repression by the process of X inactivation.
Asunto(s)
Compensación de Dosificación (Genética) , Glicoproteínas/genética , Transcripción Genética , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , Genes , Ligamiento Genético , Humanos , Células Híbridas , Metaloendopeptidasas/antagonistas & inhibidores , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Inhibidores Tisulares de MetaloproteinasasRESUMEN
We determined the expression pattern of the tissue inhibitor of metalloproteinase (TIMP) in the development of the mouse embryo using in situ hybridization and transgenesis. Localized TIMP RNA was first detected at 13.5 days post conceptus (p.c.) in tissues undergoing osteogenesis, such as the mandible, ribs, and calvaria. As development proceeded, TIMP RNA could be detected at additional sites, including the tooth buds, vertebrae, and long bones. To define the sequences regulating TIMP expression, we generated transgenic mice that expressed the Escherichia coli beta-galactosidase gene under control of a 5' region of the mouse TIMP gene containing -2158 to -58 bp upstream of the initiator ATG. By use of an in situ assay for beta-galactosidase activity, the TIMP-lacZ fusion gene product was localized to tissues that also expressed the endogenous TIMP gene, such as the mandible, calvaria, and vertebrae. The localization of TIMP to regions of intramembranous and endochondral bone is similar to that previously reported for TGF-beta, a growth modulator believed to be involved in regulation of extracellular matrix (ECM) formation. Thus, the expression of TIMP in these regions is consistent with it playing a role in ECM deposition and turnover in development.
Asunto(s)
Desarrollo Embrionario y Fetal , Regulación de la Expresión Génica , Glicoproteínas/biosíntesis , Ratones Transgénicos/genética , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Escherichia coli/genética , Glicoproteínas/genética , Ratones , Ratones Transgénicos/embriología , Odontogénesis , Especificidad de Órganos , Osteogénesis , Proteínas Recombinantes de Fusión/genética , Inhibidores Tisulares de Metaloproteinasas , beta-Galactosidasa/genéticaRESUMEN
In vivo responses to interferon (IFN) in mice were determined by measuring the steady-state levels of induced mRNAs following injection of IFN and poly(I)-poly(C). With cDNA probes for mouse 2'-5' oligoadenylate synthetase (2-5A synthetase) and 1-8, constitutive expression of the corresponding mRNA was detectable in different organs of normal C3H/He mice. These mRNA levels were increased by as much as 15-fold over control levels in various tissues, including the brain, after IFN and poly(I)-poly(C) treatment, coincident with increases in 2-5A synthetase enzyme activity. The basal activity level of this enzyme could be reduced in normal mice by treatment with anti-mouse IFN (alpha + beta) antibody. This treatment also reduced the levels of 2-5A synthetase and 1-8 mRNAs. Thus, physiological levels of circulating IFN maintain elevated levels of IFN-induced mRNAs in mice. Furthermore, changes in 2-5A synthetase enzyme activity reflect the changes in gene expression in vivo.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , Regulación de la Expresión Génica , Interferón Tipo I/farmacología , ARN Mensajero/análisis , 2',5'-Oligoadenilato Sintetasa/biosíntesis , Animales , Femenino , Ratones , Ratones Endogámicos C3H , Hibridación de Ácido Nucleico , Poli I-C/farmacología , ARN Mensajero/genética , Organismos Libres de Patógenos EspecíficosRESUMEN
Histone H1 subtype complexity and H1 histone subtype synthesis switches were characterized during the development of normal embryos of the mud snail Ilyanassa obsoleta. The effect of the removal of the third polar lobe on the normal H1 pattern of synthesis was then investigated in the delobed embryo to determine if classical polar lobe effects are accompanied by a perturbation of these patterns. SDS-gel electrophoresis and fluorography of radiolabeled 5% perchloric acid-soluble nuclear extracts resolved six H1 proteins designated bands 1-6. Bands 1-5 migrate as a cluster of individual bands with similar mobilities. Band 6 has a substantially slower mobility. The synthesis of band 6 is predominant during the first 6 hr post-trefoil. During cleavage and gastrulation bands 1 and 2 are predominant while band 3, 4, and 5 become predominant during organogenesis. In addition, it has been found that removal of the polar lobe delays the off-switch of the early bands 6, 1, and 2 and the on-switch of the late bands 3, 4, and 5. This must result in a different H1 composition in the chromatin of the two embryo types. Cell number data of normal and delobed embryos reveal that the delay in subtype synthesis switching is not caused by an overall delay of cell division in the delobed embryo. However, the data indicate that a subpopulation of cells may not divide, or may divide late, in the delayed embryo. The data also suggest that the D cell lineage may be involved in the control of histone synthesis switching in the A, B, and C cell lineages.