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Biomedical subjects

D M Ornitz

Publications and source records attributed to D M Ornitz.

At least 37 records · Page 2Linked to original sources
PubMed1999

Fibroblast growth factor receptor 3 gene transcription is suppressed by cyclic adenosine 3',5'-monophosphate. Identification of a chondrocytic regulatory element.

Signaling through fibroblast growth factor receptors (FGFRs) is critical for the development and patterning of the vertebrate skeleton. Gain-of-function alleles of fgfr2 and fgfr3 have been linked to several dominant skeletal disorders in humans, while null mutations in fgfr3 result in the overgrowth of long bones in a mouse model system. Interestingly, the expression pattern of fgfr3 in growth plate chondrocytes overlaps that of the parathyroid hormone (PTH)-related peptide (PTHrP) receptor, a signaling molecule that also regulates endochondral ossification. The coincident expression of these two receptors suggests that their signaling pathways may also interact. To gain insight into the regulatory mechanism(s) that govern the expression of the fgfr3 gene in chondrocytes, we have identified a cell-specific transcriptional regulatory element (CSRh) by measuring the activity of various promoter fragments in FGFR3-expressing (CFK2) and nonexpressing (RCJ) chondrocyte-like cell lines. Furthermore, we demonstrate that activation of PTH/PTHrP receptors, either by stimulation with PTH or through the introduction of activating mutations, represses CSRh-mediated transcriptional activity. Finally, the transcriptional repression of the CSRh element was mimicked by treatment with forskolin, 8-bromo-cAMP, and 3-isobutyl-1-methylxanthine or by overexpression of the catalytic subunit of protein kinase A. Together, these data suggest that protein kinase A activity is a critical factor that regulates fgfr3 gene expression in the proliferative or prehypertrophic compartment of the epiphyseal growth plate. Furthermore, these results provide a possible link between PTHrP signaling and fgfr3 gene expression during the process of endochondral ossification.

1-Methyl-3-isobutylxanthine↗
PubMed1999

The mouse SLIT family: secreted ligands for ROBO expressed in patterns that suggest a role in morphogenesis and axon guidance.

The Slit gene encodes a secreted molecule essential for neural development in Drosophila embryos. Here we report the identification of three Slit homologues in the mouse. We demonstrate that the mouse SLIT1 protein can bind ROBO1, a transmembrane receptor implicated in axon guidance. Both whole-mount and section in situ hybridization studies reveal unique and complementary patterns of expression of the three mouse Slit genes and of Robo1, both within the central nervous system and in other developing tissues. The complementary expression patterns of Slit and Robo1 and their in vitro interaction suggest a ligand-receptor relationship. The expression of all three Slit genes in the floor plate suggests that they are likely to share the same functional properties with their Drosophila homologue in midline neural development and axon guidance. The complementary expression of Slit and Robo1 in different subdivisions of the somites suggests their possible function in axon pathfinding and neural crest cell migration. The unique expression pattern in limb and other organs indicates additional potential functions of the Slit gene family.

Amino Acid Sequence↗
PubMed1999

Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons.

The olfactory bulb plays a central role in olfactory information processing through its connections with both peripheral and cortical structures. Axons projecting from the olfactory bulb to the telencephalon are guided by a repulsive activity in the septum. The molecular nature of the repellent is not known. We report here the isolation of vertebrate homologs of the Drosophila slit gene and show that Slit protein binds to the transmembrane protein Roundabout (Robo). Slit is expressed in the septum whereas Robo is expressed in the olfactory bulb. Functionally, Slit acts as a chemorepellent for olfactory bulb axons. These results establish a ligand-receptor relationship between two molecules important for neural development, suggest a role for Slit in olfactory bulb axon guidance, and reveal the existence of a new family of axon guidance molecules.

Animals↗
PubMed1999

Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene.

Fibroblast growth factor 9 (FGF9), originally cloned as glial-activating factor from human glioma cells, is expressed in adult rat brain and kidney. Here we report the chromosomal localization, genomic organization, and embryonic expression pattern of the mouse Fgf9 gene. Fgf9 maps to chromosome 14 near the Ctla6 locus. The gene spans more than 34 kb and contains three exons and two introns. Translation initiation occurs in exon 1, and translation termination occurs in exon 3. Fgf9 RNA was detected during mouse embryogenesis in several tissues in which Fgf gene expression has not been previously described, including intermediate mesoderm of late-stage gastrulation, ventricular myocardium, lung pleura, skeletal myoblasts in the early limb bud, spinal cord motor neurons, olfactory bulb, and gut lumenal epithelium. Fgf9 is coexpressed with other Fgf genes in some skeletal myoblasts, in limb apical ectoderm, in craniofacial ectoderm, and in the retina, inner ear, and tooth bud. Dev Dyn 1999;216:72-88.

Amino Acid Sequence↗
PubMed1999

High-resolution mapping of tlt, a mouse mutant lacking otoconia.

The ability to sense gravity is enhanced by an extracellular structure that overlies the macular sensory epithelium. This complex consists of high density particles, otoconia, embedded within a gelatinous membrane. The tilted mouse specifically lacks otoconia, yet has no other detectable anatomic lesions. Furthermore, the penetrance of the tilted phenotype is nearly 100%. This mouse provides a model to identify genes that are involved in the development and function of vestibular otoconia. Using SSLP markers, we have mapped the tilted (tlt) gene on mouse Chromosome (Chr) 5 between D5Mit421 and D5Mit353/D5Mit128/D5Mit266/D5Mit267 by analysis of the progeny of an intersubspecific F2 intercross. We also mapped the fibroblast growth factor receptor 3 (Fgfr3) gene, a potential candidate for tlt, and the Huntington's disease homolog (Hdh) gene to D5Mit268, approximately 4.3 centiMorgans (cM) from the tilted locus. This study excludes both Fgfr3 and Hdh as candidate genes for tlt and identifies closely linked microsatellite markers that will be useful for the positional cloning of tlt.

Animals↗
PubMed1999

Genomic structure, mapping, activity and expression of fibroblast growth factor 17.

Fibroblast growth factors are essential molecules for development. Here we characterize Fgfl7, a new member of the fibroblast growth factor (FGF) family. The Fgfl7 gene maps to mouse chromosome 14 and is highly conserved between mouse and human (93% identity). It exhibits 60% amino acid identity with Fgf8 and 50% identity with Fgf8. Both Fgf8 and Fgf17 have a similar structure and a similar pattern of alternative splicing in the 5' coding region. When expressed in 3T3 fibroblasts, mouse FGF17 is transforming, indicating that it can activate the 'c' splice form of either FGF receptor (FGFR) one or two. During midgestation embryogenesis, in situ hybridization analysis localized Fgf17 expression to specific sites in the midline structures of the forebrain, the midbrain-hindbrain junction, the developing skeleton and in developing arteries. Comparison to Fgf8 revealed a striking similarity in expression patterns, especially in the central nervous system (CNS), suggesting that both genes may be important for CNS development, although Fgf17 is expressed somewhat later than Fgf8. In the developing skeleton, both genes are expressed in costal cartilage while Fgf8 is preferentially expressed in long bones. In the developing great vessels Fgfl7 is preferentially expressed, suggesting that it may have a more prominent role in vascular growth.

3T3 Cells↗
PubMed1998

Otoconin-90, the mammalian otoconial matrix protein, contains two domains of homology to secretory phospholipase A2.

The ability to sense orientation relative to gravity requires dense particles, called otoconia, which are localized in the vestibular macular organs. In mammals, otoconia are composed of proteins (otoconins) and calcium carbonate crystals in a calcite lattice. Little is known about the mechanisms that regulate otoconial biosynthesis. To begin to elucidate these mechanisms, we have partially sequenced and cloned the major protein component of murine otoconia, otoconin-90 (OC90). The amino acid sequence identified an orphan chimeric human cDNA. Because of its similarity to secretory phospholipase A2 (sPLA2), this gene was referred to as PLA2-like (PLA2L) and enabled the identification of human Oc90. Partial murine cDNA and genomic clones were isolated and shown to be specifically expressed in the developing mouse otocyst. The mature mouse OC90 is composed of 453 residues and contains two domains homologous to sPLA2. The cloning of Oc90 will allow an examination of the role of this protein in otoconial biosynthesis and in diseases that affect the vestibular system.

Amino Acid Sequence↗
PubMed1998

MMTV-Fgf8 transgenic mice develop mammary and salivary gland neoplasia and ovarian stromal hyperplasia.

Prior studies have identified Fibroblast Growth Factor-8 (Fgf8) as a possible proto-oncogene in mouse mammary tumorigenesis. We now report on the generation of two types of Fgf8 transgenic mice that each utilize the mouse mammary tumor virus (MMTV) promoter. The first transgene (MMTV-Fgf8b) results in the overexpression of the FGF8b isoform exclusively. Male and female MMTV-Fgf8b transgenic mice are viable and fertile. RNA for FGF8b is detected in mammary gland and salivary gland tissues of transgenic mice by Northern blot analysis. Nearly 85% of breeding transgenic female mice developed mammary lobular adenocarcinomas by 12 months of age, while no tumors developed in non-transgenic littermates. Salivary gland tumors occurred in some animals, always in association with mammary tumors. Several MMTV-Fgf8b transgenic mice had lung metastases at necropsy. The second transgene (MMTV-Fgf8) uses the entire Fgf8 gene and potentially encodes all FGF8 isoforms. Fgf8 is expressed by this transgene in several tissues in addition to those described above, notably the ovaries. The two MMTV-Fgf8 founders developed mammary ductal adenocarcinomas at five and eight months of age, and both displayed ovarian stromal hyperplasia. The founders expressing either transgene did not successfully nurse their pups. These results demonstrate that production of FGF8b, and possibly other FGF8 isoforms, in the mammary and salivary glands contributes to oncogenesis, and that ovarian expression results in stromal hyperplasia.

Adenocarcinoma↗
PubMed1998

FGF signaling in skeletal development.

The fibroblast growth factor receptor family consists of four receptor tyrosine kinases which bind with varying affinity and specificity to a family of at least fifteen polypeptide ligands. The receptors and ligands perform many essential functions during growth, development and repair. Recent discoveries show that a growing number of skeletal abnormalities result from mutations in the fibroblast growth factor receptors. These findings have led to a greater understanding of the role of fibroblast growth factor signaling during skeletogenesis and have focused research interests on the effects of fibroblast growth factors on endochondral and intramembranous bone development.

Alternative Splicing↗
PubMed1998

Regulation of the fibroblast growth factor receptor 3 promoter and intron I enhancer by Sp1 family transcription factors.

Fibroblast growth factor receptor 3 (FGFR3) has a complex spatial and temporal pattern of expression and is essential for the normal development of a diverse set of tissues. Recently, mutations have been identified in FGFR3 that result in constitutive tyrosine kinase activity and cause a number of different human skeletal disorders. To examine the regulatory mechanisms governing FGFR3 expression, the promoter for the FGFR3 gene was identified and characterized. It resides in a CpG island, which encompasses the 5' end of the FGFR3 gene and lacks classical cis-regulatory motifs. As little as 100 base pairs of sequence 5' to the initiation site can confer a 20-40-fold increase in transcriptional activity upon a promoter-less vector. The transcriptional activity of these cis-regulatory sequences is further stimulated by elements found within the first intron. Mapping of the enhancer activity found within intron I identified two purine-rich sequence motifs between +340 and +395. Electrophoretic mobility shift assays demonstrated that sequences within this region bind members of the Sp1 family of transcription factors. In a background lacking Sp1-like activity, we demonstrate that Sp1 can enhance transcription of the minimal promoter (which contains five classical Sp1 sites), whereas both Sp1 and Sp3 can enhance transcription through the elements found in intron I. Although these transcription factors are ubiquitously expressed, we demonstrate that the sequences between -220 and +609 of the FGFR3 gene are sufficient to promote the tissue-specific expression of a reporter gene in transgenic mice.

Animals↗
PubMed1998

Otoconial agenesis in tilted mutant mice.

The sense of balance is one of the phylogenetically oldest sensory systems. The vestibular organs, consisting of sensory hair cells and an overlying extracellular membrane, have been conserved throughout vertebrate evolution. To better understand mechanisms regulating vestibular development and mechanisms of vestibular pathophysiology, we have analyzed the mouse mutant, tilted (tlt), which has dysfunction of the gravity receptors. The tilted mouse arose spontaneously and has not been previously analyzed for a developmental or physiological deficit. Here we demonstrate that the tilted mouse, like the head tilt (het) mouse, specifically lacks otoconia and consequently does not sense spatial orientation relative to the force of gravity. Unlike other mouse mutations affecting the vestibular system (such as pallid, mocha and tilted head), the defect in the tilted mouse is highly penetrant, results in the nearly complete absence of otoconia, exhibits no degeneration of the sensory epithelium and has no apparent abnormal phenotype in other organ systems. We further demonstrate that protein expression in the macular sensory epithelium is qualitatively unaltered in tilted mutant mice.

Animals↗
PubMed1998

Physiological degradation converts the soluble syndecan-1 ectodomain from an inhibitor to a potent activator of FGF-2.

The activity of fibroblast growth factor 2 (FGF-2) is stringently controlled. Inactive in undisturbed tissues, it is activated during injury and is critical for tissue repair. We find that this control can be imposed by the soluble syndecan-1 ectodomain, a heparan sulfate proteoglycan shed from cell surfaces into wound fluids. The ectodomain potently inhibits heparin-mediated FGF-2 mitogenicity because of the poorly sulfated domains in its heparin sulfate chains. Degradation of these regions by platelet heparanase produces heparin-like heparin sulfate fragments that markedly activate FGF-2 mitogenicity and are found in wound fluids. These results establish a novel physiological control for FGF-2 and suggest new ways to modulate FGF activity.

Binding Sites↗
PubMed1998

Identification and characterization of a novel member of the fibroblast growth factor family.

A new member of the fibroblast growth factor (FGF) family, FGF-13, has been molecularly cloned as a result of high throughput sequencing of a human ovarian cancer cell library. The open reading frame of the novel human gene (1419 bp) encodes for a protein of 216 a.a. with a molecular weight of 22 kDa. The FGF-13 sequence contains an amino-terminal hydrophobic region of 23 a.a. characteristic of a signal secretion sequence. FGF-13 is most homologous, 70% similarity at the amino acid level, to FGF-8. Northern hybridization analysis demonstrated prominent expression of FGF-13 in human foetal and adult brain, particularly in the cerebellum and cortex. In proliferation studies with BaF3 cells, FGF-13 preferentially activates cell clones expressing either FGF receptor variant, 3-IIIc or 4. The signal transduction pathways of FGF-13 and FGF-2 were compared in rat hippocampal astrocytes. The two FGFs induce an equivalent level of tyrosine phosphorylation of mitogen-activated protein kinase (MAPK) and c-raf activation. However, FGF-13 is more effective than FGF-2 in inducing the phosphorylation of phospholipase C-gamma (PLC-gamma). Treatment of neuronal cultures from rat embryonic cortex with FGF-13 increases the number of glutamic acid decarboxylase immunopositive neurons, the level of high-affinity gamma-aminobutyric acid (GABA) uptake, and choline acetyltransferase enzyme activity. The GABAergic neuronal response to FGF-13 treatment is rapid with a significant increase occurring within 72 h. We have identified a novel member of the FGF family that is expressed in the central nervous system (CNS) and increases the number as well as the level of phenotypic differentiation of cortical neurons in vitro.

Amino Acid Sequence↗
PubMed1998

Identification of the cytoplasmic regions of fibroblast growth factor (FGF) receptor 1 which play important roles in induction of neurite outgrowth in PC12 cells by FGF-1.

Fibroblast growth factor 1 (FGF-1) induces neurite outgrowth in PC12 cells. Recently, we have shown that the FGF receptor 1 (FGFR-1) is much more potent than FGFR-3 in induction of neurite outgrowth. To identify the cytoplasmic regions of FGFR-1 that are responsible for the induction of neurite outgrowth in PC12 cells, we took advantage of this difference and prepared receptor chimeras containing different regions of the FGFR-1 introduced into the FGFR-3 protein. The chimeric receptors were introduced into FGF-nonresponsive variant PC12 cells (fnr-PC12 cells), and their ability to mediate FGF-stimulated neurite outgrowth of the cells was assessed. The juxtamembrane (JM) and carboxy-terminal (COOH) regions of FGFR-1 were identified as conferring robust and moderate abilities, respectively, for induction of neurite outgrowth to FGFR-3. Analysis of FGF-stimulated activation of signal transduction revealed that the JM region of FGFR-1 conferred strong and sustained tyrosine phosphorylation of several cellular proteins and activation of MAP kinase. The SNT/FRS2 protein was demonstrated to be one of the cellular substrates preferentially phosphorylated by chimeras containing the JM domain of FGFR-1. SNT/FRS2 links FGF signaling to the MAP kinase pathway. Thus, the ability of FGFR-1 JM domain chimeras to induce strong sustained phosphorylation of this protein would explain the ability of these chimeras to activate MAP kinase and hence neurite outgrowth. The role of the COOH region of FGFR-1 in induction of neurite outgrowth involved the tyrosine residue at amino acid position 764, a site required for phospholipase C gamma binding and activation, whereas the JM region functioned primarily through a non-phosphotyrosine-dependent mechanism. In contrast, assessment of the chimeras in the pre-B lymphoid cell line BaF3 for FGF-1-induced mitogenesis revealed that the JM region did not play a role in this cell type. These data indicate that FGFR signaling can be regulated at the level of intracellular interactions and that signaling pathways for neurite outgrowth and mitogenesis use different regions of the FGFR.

Amino Acid Sequence↗
PubMed1998

Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3.

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of skeletal growth and activating mutations in Fgfr3 cause achondroplasia, the most common genetic form of dwarfism in humans. Little is known about the mechanism by which FGFR3 inhibits bone growth and how FGFR3 signaling interacts with other signaling pathways that regulate endochondral ossification. To understand these mechanisms, we targeted the expression of an activated FGFR3 to growth plate cartilage in mice using regulatory elements from the collagen II gene. As with humans carrying the achondroplasia mutation, the resulting transgenic mice are dwarfed, with axial, appendicular and craniofacial skeletal hypoplasia. We found that FGFR3 inhibited endochondral bone growth by markedly inhibiting chondrocyte proliferation and by slowing chondrocyte differentiation. Significantly, FGFR3 downregulated the Indian hedgehog (Ihh) signaling pathway and Bmp4 expression in both growth plate chondrocytes and in the perichondrium. Conversely, Bmp4 expression is upregulated in the perichondrium of Fgfr3-/- mice. These data support a model in which Fgfr3 is an upstream negative regulator of the hedgehog (Hh) signaling pathway. Additionally, Fgfr3 may coordinate the growth and differentiation of chondrocytes with the growth and differentiation of osteoprogenitor cells by simultaneously modulating Bmp4 and patched expression in both growth plate cartilage and in the perichondrium.

Achondroplasia↗
PubMed1998

Fibroblast growth factor receptor 2 (FGFR2)-mediated reciprocal regulation loop between FGF8 and FGF10 is essential for limb induction.

FGFR2 is a membrane-spanning tyrosine kinase that serves as a high affinity receptor for several members of the fibroblast growth factor (FGF) family. To explore functions of FGF/FGFR2 signals in development, we have mutated FGFR2 by deleting the entire immunoglobin-like domain III of the receptor. We showed that murine FGFR2 is essential for chorioallantoic fusion and placenta trophoblast cell proliferation. Fgfr2(DeltaIgIII/DeltaIgIII) embryos displayed two distinct defects that resulted in failures in formation of a functional placenta. About one third of the mutants failed to form the chorioallantoic fusion junction and the remaining mutants did not have the labyrinthine portion of the placenta. Consequently, all mutants died at 10-11 days of gestation. Interestingly, Fgfr2(DeltaIgIII/DeltaIgIII) embryos do not form limb buds. Consistent with this defect, the expression of Fgf8, an apical ectodermal factor, is absent in the mutant presumptive limb ectoderm, and the expression of Fgf10, a mesenchymally expressed limb bud initiator, is down regulated in the underlying mesoderm. These findings provide direct genetic evidence that FGF/FGFR2 signals are absolutely required for vertebrate limb induction and that an FGFR2 signal is essential for the reciprocal regulation loop between FGF8 and FGF10 during limb induction.

Animals↗
PubMed1997

Transplanted oligodendrocyte progenitor cells expressing a dominant-negative FGF receptor transgene fail to migrate in vivo.

The proliferation, migration, survival, and differentiation of oligodendrocyte progenitor cells, precursors to myelin-forming oligodendrocytes in the CNS, are controlled by a number of polypeptide growth factors in vitro. The requirement and roles for individual factors in vivo, however, are primarily unknown. We have used a cell transplantation approach to examine the role of fibroblast growth factor (FGF) in oligodendrocyte development in vivo. A dominant-negative version of the FGF receptor-1 transgene was introduced into oligodendrocyte progenitors in vitro, generating cells that were nonresponsive to FGF but responsive to other mitogens. When transplanted into the brains of neonatal rats, mutant cells were unable to migrate and remained within the ventricles. These results suggest a role for FGF signaling in establishing a motile phenotype for oligodendrocyte progenitor cell migration in vivo and illustrate the utility of a somatic cell mutagenesis approach for the study of gene function during CNS development in vivo.

Animals↗