The missense mutation W290R in Fgfr2 causes developmental defects from aberrant IIIb and IIIc signaling.

Missense mutations in the Fibroblast Growth Factor Receptor 2 (FGFR2) have been identified in human craniosynostotic syndromes such as Crouzon (CS) and Pfeiffer (PS). FGFR2 has two major isoforms, IIIb and IIIc, generated through alternative splicing with their own temporal, spatial, and ligand-binding specificities. In this study, we report the identification and characterization of a missense mutation in codon 290 of murine Fgfr2 (W290R). The defects in W290R mutants are suggestive of disruption of signalling in both IIIb and IIIc isoforms of the Fgfr2 gene. Heterozygous mutants presented with features resembling those found in patients with CS. Fgfr2(W290R) homozygotes displayed constitutive FGFR2 activation with increased, but correct tissue-specific, expression of the IIIb and IIIc isoforms in many of the defective organs. Our Fgfr2(W290R) mouse model thus represents an excellent mouse model of CS to probe the many questions around the pathogenesis of craniosynostotic birth defects consequent to defects in FGF signaling.

Segmental expression of the EphA4 (Sek-1) receptor tyrosine kinase in the hindbrain is under direct transcriptional control of Krox-20.

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres (r) with distinct identities. Recent studies have uncovered regulatory links between transcription factors governing this process, but little is known of how these relate to molecules mediating cell-cell signalling. The Eph receptor tyrosine kinase gene EphA4 (Sek-1) is expressed in r3 and r5, and function-blocking experiments suggest that it is involved in restricting intermingling of cells between odd- and even-numbered rhombomeres. We have analysed the cis-acting regulatory sequences of the EphA4 gene in transgenic mice and identified a 470 bp enhancer element that drives specific expression in r3 and r5. Within this element, we have identified eight binding sites for the Krox-20 transcription factor that is also expressed in r3 and r5. Mutation of these binding sites abolishes r3/r5 enhancer activity and ectopic expression of Krox-20 leads to ectopic activation of the enhancer. These data indicate that Krox-20 is a direct transcriptional activator of EphA4. Together with evidence that Krox-20 regulates Hox gene expression, our findings reveal a mechanism by which the identity and movement of cells are coupled such that sharply restricted segmental domains are generated.

Roles of Eph receptors and ephrins in neural crest pathfinding.

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.

Distinct and overlapping expression patterns of ligands for Eph-related receptor tyrosine kinases during mouse embryogenesis.

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.

Elk-L3, a novel transmembrane ligand for the Eph family of receptor tyrosine kinases, expressed in embryonic floor plate, roof plate and hindbrain segments.

The Eph family of receptor tyrosine kinases has 13 distinct members and seven ligands for these receptors have been described to date. These receptors and their ligands have been implicated in regulating neuronal axon guidance and in patterning of the developing nervous system and may also serve a patterning and compartmentalization role outside of the nervous system as well. The ligands are all membrane-attached, and this attachment appears to be crucial for their normal function; five of the known ligands are linked to the membrane via a glycosyl phosphotidylinositol (GPI) linkage, while two of the ligands are transmembrane proteins. Despite the large number of Eph family receptors and ligands, they can be divided into just two major subclasses based on their binding specificities. All the GPI-anchored ligands bind and activate one subclass of the Eph receptors (that represented by Eck) while the two transmembrane ligands bind and activate the other major subclass of receptors (represented by Elk). Here we report the identification and characterization of the third, and most divergent, member of the transmembrane group of Eph ligands, which we term Elk-L3 (Elk-related receptor ligand number 3). Elk-L3 is notable for its remarkably restricted and prominent expression in the floor plate and roof plate of the developing neural tube and its rhombomere-specific expression in the developing hindbrain. The Elk-L3 gene has been localized to mouse chromosome 11 and human chromosome 17.

Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis.

We report that the many Eph-related receptor tyrosine kinases, and their numerous membrane-bound ligands, can each be grouped into only two major specificity subclasses. Receptors in a given subclass bind most members of a corresponding ligand subclass. The physiological relevance of these groupings is suggested by viewing the collective distributions of all members of a subclass. These composite distributions, in contrast with less informative patterns seen with individual members of the family, reveal that the developing embryo is subdivided into domains defined by reciprocal and apparently mutually exclusive expression of a receptor subclass and its corresponding ligands. Receptors seem to encounter their ligands only at the interface between these domains. This reciprocal compartmentalization implicates the Eph family in the formation of spatial boundaries that may help to organize the developing body plan.

Cell-cell interactions and segmentation in the developing vertebrate hindbrain.

During development of the vertebrate hindbrain, regulatory gene expression becomes precisely restricted to specific segments. Studies at the cellular and molecular levels suggest that establishment of this precise pattern of gene expression may involve a dynamic regulation of cell identity and a restriction of cell movement across rhombomere boundaries. Candidates for mediating such interactions are several members of the Eph-related receptor tyrosine kinase (RTK) family that have segmental expression in the hindbrain. Ligands for members of this RTK family are membrane-bound, and may therefore mediate cell contact-dependent signalling. We discuss the expression patterns of Eph-related receptors in the hindbrain and the potential roles that these may play in patterning.

Expression of the Wilms' tumor suppressor gene WT1 during mouse embryogenesis.

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)

Coordinate expression of Wilms' tumor genes correlates with Wilms' tumor phenotypes.

The cloning and molecular characterization of two putative tumor genes, WT1 and WIT1, from the chromosome 11p13 region has provided a means of evaluating their role in the generation of Wilms' tumor heterogeneity. A series of 29 tumors were analyzed for WT1 and WIT1 expression by Northern blot or RNase protection analyses, and results were compared with tumor histopathology. Tumors were scored for the percentage of mesenchymal and epithelial derived tissue components. Homotypic tumors comprised blastema, tubular epithelium, and a fibroblast-like mesenchyme. In addition to these tissue components, the group of tumors designated as heterotypic also contained ectopic cell phenotypes such as muscle and squamous epithelium. The analyses suggest that heterotypic differentiation patterns occur when WT1 and WIT1 expression is low relative to normal fetal kidney. In situ hybridization using antisense RNA probes showed that WT1 and WIT1 were concordantly expressed in normal fetal kidney and in the blastema of tumors. The ratio of WT1:WIT1 expression remained relatively constant in homotypic tumors but deviated significantly in heterotypic tumors. These results suggest that expression patterns of the WT1 and WIT1 genes can be closely correlated to Wilms' tumor histopathology.

Regulation of TIMP gene expression in cell culture and during mouse embryogenesis.

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.

Localization of TIMP in cycling mouse hair.

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.

Identification of a serum- and phorbol ester-responsive element in the murine tissue inhibitor of metalloproteinase gene.

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.

X chromosome inactivation of the human TIMP gene.

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.

Developmental expression of the endogenous TIMP gene and a TIMP-lacZ fusion gene in transgenic mice.

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.

Expression of interferon-induced genes in different tissues of mice.

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.

H1 histone subtypes and subtype synthesis switches of normal and delobed embryos of Ilyanassa obsoleta.

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.

Improved carbohydrate tolerance in fibre-fed rats: studies of the chronic effect.

The effect of chronic consumption of diets containing either different sources of dietary fibre (8% guar, 8% pectin, or 8% multifibre) or low carbohydrate upon carbohydrate tolerance was examined in rats. Weight gain was significantly lower throughout the entire 28-day study period with the guar group and after 20 days with the multifibre group. When tested with a liquid meal (1 g sucrose/kg body weight) after 14 days on the diets, only the guar rats had significantly lower fasting and postprandial plasma glucose concentrations. After 28 days, the improved carbohydrate tolerance persisted in the guar rats and started to appear in the multifibre rats. Pectin and low carbohydrate diets had no effect upon either weight gain or carbohydrate tolerance. Consuming the fibre diets did not affect jejunal sucrase activities. Jejunal glucose uptake activity was significantly diminished when measured in fasting guar rats while postprandially activities were similar to controls. Jejunal Na-K-ATPase activities in fasting and postprandial guar rats were not related to changes in glucose uptake. These studies confirm that only certain types of dietary fibre improve carbohydrate tolerance and suggest that reduced weight gain and altered intestinal glucose uptake are factors involved in the chronic fibre effect.