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1.
Neuroscience ; 108(2): 183-206, 2001.
Article in English | MEDLINE | ID: mdl-11734354

ABSTRACT

Previous studies suggest that Fgf8 has a key role in regulating vertebrate development. In the rostral head of the embryonic chicken, there are increasing numbers of separate Fgf8 domains; these are present in tissues that appear to have previously expressed Otx2. As Fgf8 expression becomes established, Otx2 expression weakens, but remains in cells abutting the Fgf8 expression domain. These Fgf8 expression domains are closely associated with tissues expressing Bmp4 and Shh. Based on analogy with the embryonic limb, we suggest that Fgf8, Bmp4 and Shh function together in patterning regions of the embryonic head. Gene expression changes are particularly prominent in 14-21 somite stage embryos in the rostral forebrain, during early morphogenesis of the telencephalic and optic vesicles, when several new interfaces of Fgf8, Bmp4 and Shh are generated. To gain insights into the functions of fibroblast growth factor 8 (FGF8) in the embryonic forebrain, we studied the effects of implanting beads containing this protein in the dorsal prosencephalon of embryonic day 2 chicken embryos. Ectopic FGF8 had profound effects on morphogenesis of the telencephalic and optic vesicles. It disrupted formation of the optic stalk and caused a transformation of the pigment epithelium into neural retina. Within the telencephalon, FGF8 beads frequently induced a sulcus that had features of an ectopic rostral midline. The sulcus separated the telencephalon into rostral and caudal vesicles. Furthermore, we present evidence that FGF8 can regulate regionalization of the prosencephalon through inhibition of Otx2 and Emx2 expression. Thus, these experiments provide evidence that FGF8 can regulate both morphogenesis and patterning of the rostral prosencephalon (telencephalic and optic vesicles). FGF8 beads can induce midline properties (e.g. a sulcus) and can modulate the specification and differentiation of adjacent tissues. We suggest that some of these effects are through regulating the expression of homeobox genes (Otx2 and Emx2) that are known to participate in forebrain patterning.


Subject(s)
Body Patterning/genetics , Bone Morphogenetic Proteins/genetics , Eye/metabolism , Fibroblast Growth Factors/genetics , Gene Expression Regulation, Developmental/physiology , Nerve Tissue Proteins/genetics , Telencephalon/metabolism , Trans-Activators/genetics , Age Factors , Animals , Apoptosis/drug effects , Apoptosis/physiology , Body Patterning/drug effects , Bone Morphogenetic Protein 4 , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Division/drug effects , Cell Division/physiology , Chick Embryo , Embryonic Induction/drug effects , Embryonic Induction/physiology , Eye/drug effects , Eye/embryology , Eye Abnormalities/chemically induced , Eye Abnormalities/genetics , Eye Abnormalities/metabolism , Fibroblast Growth Factor 8 , Fibroblast Growth Factors/pharmacology , Gastrula/cytology , Gastrula/drug effects , Gastrula/metabolism , Gene Expression Regulation, Developmental/drug effects , Head/embryology , Hedgehog Proteins , Homeodomain Proteins/genetics , Otx Transcription Factors , RNA, Messenger/metabolism , Telencephalon/abnormalities , Telencephalon/drug effects , Transcription Factors
2.
Development ; 126(6): 1189-200, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10021338

ABSTRACT

Beads containing recombinant FGF8 (FGF8-beads) were implanted in the prospective caudal diencephalon or midbrain of chick embryos at stages 9-12. This induced the neuroepithelium rostral and caudal to the FGF8-bead to form two ectopic, mirror-image midbrains. Furthermore, cells in direct contact with the bead formed an outgrowth that protruded laterally from the neural tube. Tissue within such lateral outgrowths developed proximally into isthmic nuclei and distally into a cerebellum-like structure. These morphogenetic effects were apparently due to FGF8-mediated changes in gene expression in the vicinity of the bead, including a repressive effect on Otx2 and an inductive effect on En1, Fgf8 and Wnt1 expression. The ectopic Fgf8 and Wnt1 expression domains formed nearly complete concentric rings around the FGF8-bead, with the Wnt1 ring outermost. These observations suggest that FGF8 induces the formation of a ring-like ectopic signaling center (organizer) in the lateral wall of the brain, similar to the one that normally encircles the neural tube at the isthmic constriction, which is located at the boundary between the prospective midbrain and hindbrain. This ectopic isthmic organizer apparently sends long-range patterning signals both rostrally and caudally, resulting in the development of the two ectopic midbrains. Interestingly, our data suggest that these inductive signals spread readily in a caudal direction, but are inhibited from spreading rostrally across diencephalic neuromere boundaries. These results provide insights into the mechanism by which FGF8 induces an ectopic organizer and suggest that a negative feedback loop between Fgf8 and Otx2 plays a key role in patterning the midbrain and anterior hindbrain.


Subject(s)
Body Patterning/drug effects , Brain Stem/embryology , Cerebellum/embryology , Embryonic Induction , Fibroblast Growth Factors/pharmacology , Nerve Tissue Proteins/biosynthesis , Trans-Activators/biosynthesis , Zebrafish Proteins , Animals , Brain Stem/drug effects , Cerebellum/drug effects , Chick Embryo , Fibroblast Growth Factor 8 , Gene Expression Regulation, Developmental , Homeodomain Proteins/biosynthesis , Mesencephalon/embryology , Morphogenesis , Otx Transcription Factors , Proto-Oncogene Proteins/biosynthesis , Rhombencephalon/embryology , Wnt Proteins , Wnt1 Protein
3.
Development ; 125(13): 2381-95, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9609821

ABSTRACT

We describe the isolation of zebrafish Fgf8 and its expression during gastrulation, somitogenesis, fin bud and early brain development. By demonstrating genetic linkage and by analysing the structure of the Fgf8 gene, we show that acerebellar is a zebrafish Fgf8 mutation that may inactivate Fgf8 function. Homozygous acerebellar embryos lack a cerebellum and the midbrain-hindbrain boundary organizer. Fgf8 function is required to maintain, but not initiate, expression of Pax2.1 and other marker genes in this area. We show that Fgf8 and Pax2.1 are activated in adjacent domains that only later become overlapping, and activation of Fgf8 occurs normally in no isthmus embryos that are mutant for Pax2.1. These findings suggest that multiple signaling pathways are independently activated in the midbrain-hindbrain boundary primordium during gastrulation, and that Fgf8 functions later during somitogenesis to polarize the midbrain. Fgf8 is also expressed in a dorsoventral gradient during gastrulation and ectopically expressed Fgf8 can dorsalize embryos. Nevertheless, acerebellar mutants show only mild dorsoventral patterning defects. Also, in spite of the prominent role suggested for Fgf8 in limb development, the pectoral fins are largely unaffected in the mutants. Fgf8 is therefore required in development of several important signaling centers in the zebrafish embryo, but may be redundant or dispensable for others.


Subject(s)
Body Patterning/genetics , Brain/embryology , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/physiology , Amino Acid Sequence , Animals , Blastoderm/physiology , Brain/abnormalities , Cerebellum/abnormalities , Chickens , Cloning, Molecular , Crosses, Genetic , Embryonic Induction/genetics , Fibroblast Growth Factor 8 , Fibroblast Growth Factors/deficiency , Gastrula/physiology , Gene Expression Regulation, Developmental , Heterozygote , Humans , Mice , Molecular Sequence Data , Mutation , RNA Probes , Sequence Alignment , Sequence Homology, Amino Acid , Somites , Zebrafish
4.
Genomics ; 36(1): 39-46, 1996 Aug 15.
Article in English | MEDLINE | ID: mdl-8812414

ABSTRACT

We have generated a YAC contig of at least 3.3 Mb from the proximal region of In(17)4 of mouse chromosome 17. This region corresponds to DNA lost in the gastrulation mutant tw18, which belongs to the tcl-4 complementation group. Our most proximal and distal probes lie within the deletion-3.3 Mb apart-indicating that we have not cloned the entire region. The deleted region is contained in a genetic interval of less than 1 cM, suggesting that some suppression of recombination must occur.


Subject(s)
Alleles , Chromosome Deletion , Embryonic and Fetal Development/genetics , Gastrula , Animals , Chromosome Walking , Chromosomes, Artificial, Yeast/genetics , DNA Probes , Genetic Complementation Test , Genetic Markers , Mice , Mice, Inbred C57BL , Recombination, Genetic , Restriction Mapping
5.
Nature ; 380(6569): 66-8, 1996 Mar 07.
Article in English | MEDLINE | ID: mdl-8598907

ABSTRACT

Vertebrate midbrain development depends on an organizing centre located at the isthmus, a constriction in the embryonic mid/hindbrain region. Isthmic tissue grafts transform chick caudal forebrain into an ectopic midbrain that is the mirror image of the normal midbrain. Here we report that FGF8 protein has the same midbrain-inducing and polarizing effect as isthmic tissue. Moreover, FGF8 induces ectopic expression in the forebrain of genes normally expressed in the isthmus, suggesting that the ectopic midbrain forms under the influence of signals from a new 'isthmus-like' organizing centre induced in the forebrain. Because Fgf8 itself is expressed in the isthmus, our results identify FGF8 as an important signalling molecule in normal midbrain development.


Subject(s)
Fibroblast Growth Factors , Growth Substances/physiology , Mesencephalon/embryology , Neoplasm Proteins/physiology , Zebrafish Proteins , Animals , Chick Embryo , Embryonic Induction , Fibroblast Growth Factor 8 , Gene Expression Regulation, Developmental , Growth Substances/genetics , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Mesencephalon/cytology , Microspheres , Neoplasm Proteins/genetics , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Proto-Oncogene Proteins/biosynthesis , Wnt Proteins
6.
Cell ; 84(1): 127-36, 1996 Jan 12.
Article in English | MEDLINE | ID: mdl-8548816

ABSTRACT

We provide evidence that FGF8 serves as an endogenous inducer of chick limb formation and that its expression in the intermediate mesoderm at the appropriate time and place to trigger forelimb development is directly linked to the mechanism of embryonic kidney differentiation. One function of the limb inducer is to initiate Fgf8 gene expression in the ectoderm overlying the prospective limb-forming territories. FGF8 secreted by the ectoderm then appears to initiate limb bud formation by promoting outgrowth of and Sonic hedgehog expression in the underlying lateral plate mesoderm. FGF8 also maintains mesoderm outgrowth and Sonic hedgehog expression in the established limb bud. Our data thus point to FGF8 as a key regulator of limb development that not only induces and initiates the formation of a limb bud, but also sustains its subsequent development.


Subject(s)
Embryonic Induction/physiology , Extremities/embryology , Fibroblast Growth Factors , Growth Substances/physiology , Neoplasm Proteins/physiology , Trans-Activators , Amino Acid Sequence , Animals , Chick Embryo , DNA, Complementary/analysis , Ectoderm/physiology , Fibroblast Growth Factor 8 , Gene Expression Regulation, Developmental/physiology , Growth Substances/genetics , Hedgehog Proteins , Mesoderm/physiology , Molecular Sequence Data , Neoplasm Proteins/genetics , Proteins/physiology
7.
Proc Natl Acad Sci U S A ; 92(13): 5870-4, 1995 Jun 20.
Article in English | MEDLINE | ID: mdl-7597044

ABSTRACT

To identify genes involved in the regulation of early mammalian development, we have developed a dominant-negative mutant basic-helix-loop-helix (bHLH) protein probe for interaction cloning and have isolated a member of the bHLH family of transcription factors, Meso1. Meso1-E2A heterodimers are capable of binding to oligonucleotide probes that contain a bHLH DNA recognition motif. In mouse embryos, Meso1 is expressed prior to MyoD1 family members. Meso1 expression is first detected at the neural plate stage of development in the paraxial mesoderm of the head and in presomitic mesodermal cells prior to their condensation into somites. Our findings suggest that Meso1 may be a key regulatory gene involved in the early events of vertebrate mesoderm differentiation.


Subject(s)
Chromosome Mapping , DNA-Binding Proteins/biosynthesis , Mesoderm/physiology , Transcription Factors/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors , Cloning, Molecular , Crosses, Genetic , DNA Primers , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Embryonic and Fetal Development , Female , Helix-Loop-Helix Motifs , In Situ Hybridization , Kidney/metabolism , Male , Mesoderm/cytology , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Muridae , Muscle, Skeletal/metabolism , MyoD Protein/biosynthesis , MyoD Protein/genetics , Myocardium/metabolism , Organ Specificity , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Testis/metabolism , Trans-Activators/biosynthesis , Transcription Factors/genetics , Transcription Factors/physiology
8.
Development ; 121(2): 439-51, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7768185

ABSTRACT

Evidence is accumulating that members of the FGF gene family provide signals that act locally to regulate growth and patterning in vertebrate embryos. In this report, we provide a detailed analysis of the mouse Fgf8 gene. We have mapped the Fgf8 locus to the distal region of mouse chromosome 19, and sequenced the 5' coding region of the gene. Our data identify a new coding exon, and locate multiple splice donor and splice acceptor sites that can be used to produce at least seven transcripts encoding a family of secreted FGF8 proteins with different N termini. From these results, it appears that Fgf8 is structurally the most complex member of the FGF family described to date. In the embryo, many of the regions in which Fgf8 RNA is localized are known to direct outgrowth and patterning, including the apical ectodermal ridge of the limb bud, the primitive streak and tail bud, the surface ectoderm overlying the facial primorida and the midbrain-hindbrain junction, suggesting that FGF8 may be a component of the regulatory signals that emanate from these regions.


Subject(s)
Fibroblast Growth Factors/genetics , Gastrula/physiology , Amino Acid Sequence , Animals , Brain/embryology , Chromosome Mapping , DNA Primers/genetics , Extremities/embryology , Gene Expression , Genome , In Situ Hybridization , Mice , Mice, Transgenic , Molecular Sequence Data , Morphogenesis/genetics , Tail/embryology
9.
Development ; 119(2): 295-306, 1993 Oct.
Article in English | MEDLINE | ID: mdl-7507029

ABSTRACT

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.


Subject(s)
Carrier Proteins/genetics , Cell Adhesion Molecules/genetics , Morphogenesis/genetics , Receptors, Cell Surface/genetics , Receptors, Lymphocyte Homing/genetics , Animals , Carrier Proteins/physiology , Embryo, Mammalian/metabolism , Epithelium/physiology , Extremities/embryology , Gene Expression/physiology , Heart/embryology , Hyaluronan Receptors , Hyaluronic Acid/metabolism , Immunoblotting , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Inbred Strains , Odontogenesis , Receptors, Cell Surface/physiology , Receptors, Lymphocyte Homing/physiology
10.
Cancer Res ; 52(5): 1137-43, 1992 Mar 01.
Article in English | MEDLINE | ID: mdl-1737372

ABSTRACT

We describe the characterization of human lymphoblastoid cell lines with acquired resistance (greater than 20,000-fold) to a novel folate-based thymidylate synthase (TS) (EC 2.1.1.45) inhibitor, C2-desamino-C2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI198583). This acquired resistance was associated with a 64-fold amplification of the TS gene, a similar elevation in the corresponding mRNA, and an approximately 200-fold increase in both TS activity and TS protein. This amplification was maintained when the cells were grown in the absence of the selective agent, ICI198583, for 340 generations. TS isolated from one of the resistant cell lines, W1-L2:C1, displayed inhibition kinetic parameters similar to those of TS isolated from the parent W1-L2 cell line. It thus appears unlikely that resistance is due to an altered TS enzyme having a lower affinity for ICI198583. The resistant cell line, W1-L2:C1, was cross-resistant to other folate-based TS inhibitors but was as sensitive as the parent cell line, W1-L2, to 5-fluorodeoxyuridine. The W1-L2:C1 cell line was collaterally sensitive to the classical dihydrofolate reductase (EC 1.5.1.3) inhibitor methotrexate as well as to the lipophilic dihydrofolate reductase inhibitors metoprine and 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazolin e glucuronic acid salt (also called trimetrexate). When the W1-L2 and W1-L2:C1 cell lines were exposed to 1 microM ICI198583 for 24 h they accumulated the same concentration of total cellular ICI198583 polyglutamates despite the fact that the latter cell line accumulated a 300-fold greater concentration of ICI198583 monoglutamate. As polyglutamates, the tetra- and pentaglutamate forms predominated in the W1-L2 cell line, whereas the diglutamate form predominated in the W1-L2:C1 cell line, with few higher polyglutamates being detected. The lack of tri- and higher polyglutamates of ICI198583 (i.e., the more active species) in the W1-L2:C1 cell line may also contribute to the observed resistance. These findings may have important implications in light of the rapid onset of resistance to antifolates in the clinic.


Subject(s)
Antineoplastic Agents/pharmacology , Folic Acid/analogs & derivatives , Lymphocytes/drug effects , Thymidylate Synthase/metabolism , Antineoplastic Agents/metabolism , Cell Line , Drug Resistance , Folic Acid/metabolism , Folic Acid/pharmacology , Humans , Lymphocytes/enzymology , Thymidylate Synthase/antagonists & inhibitors , Thymidylate Synthase/genetics
11.
Proc Natl Acad Sci U S A ; 88(18): 7923-7, 1991 Sep 15.
Article in English | MEDLINE | ID: mdl-1680234

ABSTRACT

We report that a number of related zinc finger protein genes are closely linked on mouse chromosome 17. At least four of these genes are transcribed in the 8.5-day postcoitum embryo and are deleted in the t complex early acting embryonic lethal mutation tw18. We have evidence that additional finger protein genes are located in this region. These findings demonstrate that related finger protein genes can be clustered in the murine genome and identify genes that may be considered as candidates for the tw18 mutation.


Subject(s)
Genes, Lethal , Zinc Fingers , Base Sequence , Chromosome Deletion , Chromosome Mapping , Cloning, Molecular , DNA/genetics , DNA Probes , Genes , Molecular Sequence Data , Oligonucleotides/chemistry , Polymorphism, Restriction Fragment Length , Sequence Homology, Nucleic Acid
12.
J Biol Chem ; 262(15): 7368-73, 1987 May 25.
Article in English | MEDLINE | ID: mdl-3584117

ABSTRACT

We report that the gene for thymidylate synthase (TS) is amplified in the mouse cell line L1210:C15 that was selectively grown in increasing concentrations of the competitive inhibitor of thymidylate synthase, CB3717. The gene is amplified 50-fold compared to the parental cell line. Amplification has not been accompanied by any major rearrangements, and the increase in gene copy number is reflected in elevation of thymidylate synthase mRNA levels. The amplification is relatively stable as there was only a 2- to 3-fold decrease in the number of amplified TS genes when cells were grown in the absence of selection for 375 generations. We also observe a 30- to 40-fold increase in number of copies of the dihydrofolate reductase gene with 7-fold elevation of the RNA product, and we suggest that this may be due to cross-inhibition of dihydrofolate reductase by CB3717. Thymidylate synthase mRNA levels in L1210 and L1210:C15 show no variation within the different phases of the cell cycle but are significantly reduced during quiescence.


Subject(s)
Cell Cycle , Gene Amplification , RNA, Messenger/metabolism , Thymidylate Synthase/genetics , Animals , Cell Line , DNA/genetics , Folic Acid/analogs & derivatives , Folic Acid/pharmacology , Folic Acid Antagonists , Leukemia L1210 , Mice , Nucleic Acid Hybridization , Quinazolines/pharmacology , Tetrahydrofolate Dehydrogenase/genetics , Thymidylate Synthase/antagonists & inhibitors
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