RÉSUMÉ
Here we demonstrate the feasibility of a doubly regulatable transgenic mouse design that allows for gene manipulation by both Cre-recombinase and the tetracycline inducible system. Using a knock-in strategy to insert both elements of the tetracycline inducible system and a neomycin (neo) cassette flanked by loxP sequences (floxed) into the wild-type locus, we generated mice that express the 5-HT(1B) receptor in a conditional manner. In the presence of a floxed neo-cassette, receptor expression was silenced. Removal of this cassette by Cre-mediated recombination led to 5-HT(1B) receptor expression, which was highly regulatable when doxycycline, a derivative of tetracycline, was administered to the mice. This system allowed for a determination of an in vivo time course of receptor half-life and recovery. Physiological studies also demonstrated that rescued 5-HT(1B) receptors were functional, and that this functionality was reversible upon treatment with doxycycline. Crossing mice where the 5-HT(1B), or the 5-HT(1A), receptors were silenced by the neo-cassette, with mice expressing either Cre-recombinase or the tetracycline transactivator (tTA) under the control of tissue-specific promoters, led to tissue-specific re-expression of these receptors. Our studies thus demonstrate the potential of this strategy for achieving both a classic knockout, as well as subsequent tissue-specific and/or inducible knockouts.
Sujet(s)
Délétion de gène , Récepteurs sérotoninergiques/génétique , Récepteurs sérotoninergiques/métabolisme , Animaux , Doxycycline/pharmacologie , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Régulation de l'expression des gènes/génétique , Période , Néomycine/pharmacologie , Récepteurs sérotoninergiques/déficitRÉSUMÉ
PURPOSE: To document the evolution of ocular motor abnormalities in an infant with carbohydrate-deficient glycoprotein syndrome. METHODS: Case report. An infant with carbohydrate-deficient glycoprotein syndrome type 1a underwent magnetic resonance imaging and infrared eye movement recording. RESULTS: A 10-month-old male with carbohydrate-deficient glycoprotein syndrome type Ia had rapid horizontal oscillations of the eyes when startled or awakened from sleep. Clinical examination confirmed this finding and disclosed congenital ocular motor apraxia with a reduced vestibulo-ocular reflex. Infrared eye movement recording showed ocular flutter and square wave jerks superimposed on a horizontal pendular nystagmus. Magnetic resonance imaging showed diffuse cerebellar hypoplasia. CONCLUSION: Carbohydrate-deficient glycoprotein syndrome type Ia can be associated with multiple cerebellar eye signs including ocular flutter, square-wave jerks, and congenital ocular motor apraxia.
Sujet(s)
Apraxies/étiologie , Cervelet/malformations , Troubles congénitaux de la glycosylation/complications , Troubles de la motilité oculaire/étiologie , Apraxies/diagnostic , Troubles congénitaux de la glycosylation/enzymologie , Mouvements oculaires , Humains , Nourrisson , Imagerie par résonance magnétique , Mâle , Nystagmus pathologique/étiologie , Troubles de la motilité oculaire/diagnostic , Phosphotransferases phosphomutases/déficit , Réflexe vestibulo-oculaireRÉSUMÉ
We report the cloning and characterization of a new member of the Delta family of Notch ligands, which we have named Dll4. Like other Delta genes, Dll4 is predicted to encode a membrane-bound ligand, characterized by an extracellular region containing several EGF-like domains and a DSL domain required for receptor binding. In situ analysis reveals a highly selective expression pattern of Dll4 within the vascular endothelium. The activity and expression of Dll4 and the known actions of other members of this family suggest a role for Dll4 in the control of endothelial cell biology.
Sujet(s)
Artères/métabolisme , Endothélium vasculaire/métabolisme , Protéines et peptides de signalisation intracellulaire/génétique , Protéines membranaires/biosynthèse , Protéines membranaires/génétique , Récepteurs de surface cellulaire , Facteurs de transcription , Protéines adaptatrices de la transduction du signal , Séquence d'acides aminés , Animaux , Séquence nucléotidique , Technique de Northern , Protéines de liaison au calcium , Cartographie chromosomique , Chromosomes humains de la paire 15 , Clonage moléculaire , ADN complémentaire/métabolisme , Humains , Hybridation in situ , Protéines et peptides de signalisation intracellulaire/composition chimique , Protéines et peptides de signalisation intracellulaire/métabolisme , Ligands , Protéines membranaires/composition chimique , Protéines membranaires/métabolisme , Souris , Données de séquences moléculaires , Liaison aux protéines , Protéines proto-oncogènes/métabolisme , Récepteur Notch1 , Récepteur Notch4 , Récepteurs Notch , Similitude de séquences d'acides aminés , Distribution tissulaire , XenopusRÉSUMÉ
The Notch gene family encodes large transmembrane receptors that are components of an evolutionarily conserved intercellular signaling mechanism. To assess the role of the Notch4 gene, we generated Notch4-deficient mice by gene targeting. Embryos homozygous for this mutation developed normally, and homozygous mutant adults were viable and fertile. However, the Notch4 mutation displayed genetic interactions with a targeted mutation of the related Notch1 gene. Embryos homozygous for mutations of both the Notch4 and Notch1 genes often displayed a more severe phenotype than Notch1 homozygous mutant embryos. Both Notch1 mutant and Notch1/Notch4 double mutant embryos displayed severe defects in angiogenic vascular remodeling. Analysis of the expression patterns of genes encoding ligands for Notch family receptors indicated that only the Dll4 gene is expressed in a pattern consistent with that expected for a gene encoding a ligand for the Notch1 and Notch4 receptors in the early embryonic vasculature. These results reveal an essential role for the Notch signaling pathway in regulating embryonic vascular morphogenesis and remodeling, and indicate that whereas the Notch4 gene is not essential during embryonic development, the Notch4 and Notch1 genes have partially overlapping roles during embryogenesis in mice.
Sujet(s)
Vaisseaux sanguins/embryologie , Protéines proto-oncogènes/physiologie , Récepteurs de surface cellulaire , Facteurs de transcription , Facteurs âges , Animaux , Embryon de mammifère/métabolisme , Homozygote , Hybridation in situ , Protéines et peptides de signalisation intracellulaire , Ligands , Protéines membranaires/biosynthèse , Protéines membranaires/génétique , Souris , Souris de lignée C57BL , Morphogenèse , Mutagenèse , Néovascularisation physiologique/génétique , Phénotype , Protéines proto-oncogènes/génétique , Récepteurs à activité tyrosine kinase/biosynthèse , Récepteur Notch1 , Récepteur Notch4 , Récepteur facteur croissance/biosynthèse , Récepteurs Notch , Récepteurs aux facteurs de croissance endothéliale vasculaire , RT-PCR , Transduction du signalRÉSUMÉ
The serotonergic system is involved in the modulation of prepulse inhibition (PPI) and habituation of startle, which are deficient in schizophrenia patients. PPI is the reduction in startle amplitude that occurs when a weak "prepulse" precedes a startling stimulus by 30-500 msec. The roles of 5-HT(1A) and 5-HT(1B) receptors in modulating PPI and habituation were examined using wild-type (WT), 5-HT(1A) knockout (1AKO), and 5-HT(1B) knockout (1BKO) mice. The 5-HT(1A/1B) agonist RU24969 reduced PPI and habituation in WT and 1AKO, but not 1BKO mice, whereas the 5-HT(1A) agonist 8-OH-DPAT increased PPI in WT and 1BKO, but not in 1AKO mice. Similarly, the selective 5-HT(1B) agonist anpirtoline reduced PPI in WT, but not in 1BKO mice. In experiments using intact 129Sv mice, the 5-HT(1A) agonist flesinoxan increased PPI while anpirtoline decreased PPI and habituation. Findings suggest that 5-HT(1B) receptor activation decreases PPI and habituation, and 5-HT(1A) receptor activation increases PPI in mice.
Sujet(s)
Inhibition nerveuse/effets des médicaments et des substances chimiques , Récepteurs sérotoninergiques/effets des médicaments et des substances chimiques , 7-Dipropylamino-5,6,7,8-tétrahydro-1-naphtol/pharmacologie , Animaux , Encéphale/effets des médicaments et des substances chimiques , Encéphale/physiologie , Femelle , Habituation/effets des médicaments et des substances chimiques , Habituation/physiologie , Indoles/pharmacologie , Souris , Souris knockout , Pipérazines/pharmacologie , Pipéridines/pharmacologie , Pyridines/pharmacologie , Récepteur de la sérotonine de type 5-HT1B , Récepteurs de la sérotonine de type 5-HT1 , Réflexe de sursaut/effets des médicaments et des substances chimiques , Réflexe de sursaut/physiologie , Agonistes des récepteurs de la sérotonine/pharmacologieRÉSUMÉ
BACKGROUND: The probability of visual recovery in tumor-related optic neuropathy usually correlates with the severity and duration of optic pathway compromise. Recovery of visual acuity to normal levels is unexpected after profound loss of vision extending for a period of weeks and months. METHODS: A 9-year-old girl who had neurosurgical resection of a craniopharyngioma compressing the optic chiasm and optic tract was followed up serially with neuroimaging and clinical examinations over a 6-year period. RESULTS: Within 3 months of the diagnosis of craniopharyngioma, the girl's vision was reduced to no-light-perception blindness when she viewed with the more involved eye. The blindness correlated with an amaurotic (i.e., >3.6 log unit) relative afferent pupillary defect and an absence of any response when tested with visual field perimetry. After more than a year of total blindness and cessation of all neurosurgical and radiation therapy, visual acuity recovered to a normal level (20/25), the afferent pupillary defect improved, and sensitivity in a portion of the temporal hemivisual field was restored. In the follow-up that has extended for 5 years from the time of recovery, stability of the restored vision has been documented. CONCLUSION: Children who have tumor-related loss of vision due to damage to the anterior visual pathways may be capable of recovery after intervals of blindness that would be considered irreversible in adults. The mechanism of the recovery in our patient may have been decompression-related restoration of axoplasmic flow, followed by gradual remyelination of visual fibers, which allowed reorganization of connections to the lateral geniculate nucleus to optimize synaptic transmission.
Sujet(s)
Cécité/physiopathologie , Craniopharyngiome/complications , Tumeurs de l'hypophyse/complications , Acuité visuelle , Cécité/étiologie , Cécité/thérapie , Encéphale/anatomopathologie , Enfant , Craniopharyngiome/diagnostic , Craniopharyngiome/chirurgie , Diagnostic différentiel , Femelle , Humains , Imagerie par résonance magnétique , Syndromes de compression nerveuse/étiologie , Syndromes de compression nerveuse/anatomopathologie , Syndromes de compression nerveuse/physiopathologie , Chiasma optique/anatomopathologie , Chiasma optique/physiopathologie , Atteintes du nerf optique/étiologie , Atteintes du nerf optique/anatomopathologie , Atteintes du nerf optique/physiopathologie , Tumeurs de l'hypophyse/diagnostic , Tumeurs de l'hypophyse/chirurgie , Champs visuels , Voies optiques/physiopathologieRÉSUMÉ
We have assigned the disulfide structure of Md-65 agouti-related protein (Md65-AGRP) using differential reduction and alkylation followed by direct sequencing analysis. The mature human AGRP is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The C-terminal domain, a 48 amino acid peptide named Md65-AGRP, was expressed in Escherichia coil cells and refolded under different conditions from the mature recombinant protein. The disulfide bonds in the cystine knot structure of Md65-AGRP were partially reduced using tris(2-carboxyethyl) phosphine (TCEP) under acidic conditions, followed by alkylation with N-ethylmaleimide (NEM). The procedure generated several isoforms with varying degrees of NEM alkylation. The multiple forms of Md65-AGRP generated by partial reduction and NEM modification were then completely reduced and carboxymethylated to identify unreactive disulfide bonds. Differentially labeled Md65-AGRP were directly sequenced and analyzed by MALDI mass spectrometry. The results confirmed that Md65-AGRP contained the same disulfide structure as that of Md5-AGRP reported previously [Bures, E. J., Hui, J. O., Young, Y. et al. (1998) Biochemistry 37, 12172-12177].
Sujet(s)
Cystéine/composition chimique , Disulfures/composition chimique , Protéines/composition chimique , Protéine apparentée à Agouti , Chromatographie en phase liquide à haute performance , N-Éthyl-maléimide/composition chimique , Humains , Protéines et peptides de signalisation intercellulaire , Cartographie peptidique , Protéines recombinantes/composition chimique , Spectrométrie de masse MALDIRÉSUMÉ
The 5-HT1A and 5-HT1B serotonin receptors are expressed in a variety of neurons in the central nervous system. While the 5-HT1A receptor is found on somas and dendrites, the 5-HT1B receptor has been suggested to be localized predominantly on axon terminals. To study the intracellular addressing of these receptors, we have used in vitro systems including Madin-Darby canine kidney (MDCK II) epithelial cells and primary neuronal cultures. Furthermore, we have extended these studies to examine addressing in vivo in transgenic mice. In epithelial cells, 5-HT1A receptors are found on both apical and basolateral membranes while 5-HT1B receptors are found exclusively in intracellular vesicles. In hippocampal neuronal cultures, 5-HT1A receptors are expressed on somatodendritic membranes but are absent from axons. In contrast, 5-HT1B receptors are found on both dendritic and axonal membranes, including growth cones where they accumulate. Using 5-HT1A and 5-HT1B knockout mice and the binary tTA/tetO system, we generated mice expressing these receptors in striatal neurons. These in vivo experiments demonstrate that, in striatal medium spiny neurons, the 5-HT1A receptor is restricted to the somatodendritic level, while 5-HT1B receptors are shipped exclusively toward axon terminals. Therefore, in all systems we have examined, there is a differential sorting of the 5-HT1A and 5-HT1B receptors. Furthermore, we conclude that our in vivo transgenic system is the only model that reconstitutes proper sorting of these receptors.
Sujet(s)
Encéphale/physiologie , Cellules épithéliales/physiologie , Neurones/physiologie , Récepteurs sérotoninergiques/physiologie , 7-Dipropylamino-5,6,7,8-tétrahydro-1-naphtol/pharmacocinétique , Animaux , Autoradiographie , Lignée cellulaire , Membrane cellulaire/physiologie , Membrane cellulaire/ultrastructure , Cellules cultivées , Corps strié/physiologie , Chiens , Cellules épithéliales/ultrastructure , Iodocyanopindolol/pharmacocinétique , Rein , Souris , Souris knockout , Souris transgéniques , Microscopie immunoélectronique , Neurones/ultrastructure , Dosage par compétition , Récepteur de la sérotonine de type 5-HT1B , Récepteurs sérotoninergiques/analyse , Récepteurs sérotoninergiques/génétique , Récepteurs de la sérotonine de type 5-HT1 , Protéines recombinantes/biosynthèse , TransfectionRÉSUMÉ
The agouti-related protein gene (Agrp) is a novel gene implicated in the control of feeding behavior. The hypothalamic expression of Agrp is regulated by leptin, and overexpression of Agrp in transgenic animals results in obesity and diabetes. By analogy with the known actions of agouti, these data suggest a role for the Agrp gene product in the regulation of melanocortin receptors expressed in the central nervous system. The availability of recombinant, highly purified protein is required to fully address this potential interaction. A nearly full-length form of AGRP (MKd5-AGRP) was expressed in the cytosolic or soluble fraction of Escherichia coli and appeared as large intermolecular disulfide-bonded aggregates. Following oxidation, refolding, and purification, this protein was soluble, and eluted as a single symmetric peak on RP-HPLC. Circular dichroism studies indicated that the purified protein contains primarily random coil and beta-sheet secondary structure. Sedimentation velocity studies at neutral pH demonstrated that MKd5-AGRP is monomeric at low micromolar concentrations. Mobility shifts observed using SDS-PAGE under reducing and nonreducing conditions for bacterially expressed and mammalian expressed AGRP were identical, an indication of a similar disulfide structure. The purification to homogeneity of a second, truncated form of AGRP (Md65-AGRP) which was expressed in the insoluble or inclusion body fraction is also described. Both forms act as competitive antagonists of alpha-melanocyte stimulating hormone (alpha-MSH) at melanocortin-3 (MC-3) and melanocortin-4 receptors (MC-4). The demonstration that AGRP is an endogenous antagonist with respect to these receptors is a unique mechanism within the central nervous system, and has important implications in the control of feeding.
Sujet(s)
Protéines et peptides de signalisation intercellulaire , Protéines/composition chimique , Protéines/pharmacologie , Protéines recombinantes/composition chimique , Protéines recombinantes/pharmacologie , Protéine de signalisation Agouti , Protéine apparentée à Agouti , Séquence d'acides aminés , Lignée cellulaire , Phénomènes chimiques , Chimie physique , Dichroïsme circulaire , Escherichia coli/métabolisme , Vecteurs génétiques/synthèse chimique , Vecteurs génétiques/métabolisme , Humains , Rein , Lysine/génétique , Méthionine/génétique , Données de séquences moléculaires , Oxydoréduction , Adénohypophyse , Liaison aux protéines/génétique , Pliage des protéines , Protéines/génétique , Protéines recombinantes/isolement et purification , Spectrométrie de masse MALDI , Thiols/analyse , TransfectionRÉSUMÉ
The agouti-related protein gene (Agrp) plays an important role in body weight regulation. The mature human protein is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The disulfide structure of recombinant human AGRP was determined by chemical methods using partial reduction with tris(2-carboxyethyl)phosphine under acidic conditions, followed by direct alkylation with N-ethylmaleimide or fluorescein-5-maleimide. Partial reduction and alkylation provided several forms of AGRP that were modified in a stepwise fashion. The resulting proteins were characterized by peptide mapping, sequence analysis, and mass spectrometry, showing that AGRP contained a highly reducible disulfide bond, C85-C109, followed by less reactive ones, C90-C97, C74-C88, C67-C82, and C81-C99, respectively. The chemically defined disulfide connectivity of the recombinant human AGRP was homologous to that of omega-agatoxin IVB except for an additional disulfide bond, C85-C109.
Sujet(s)
Disulfures/composition chimique , Protéines et peptides de signalisation intercellulaire , Protéines/composition chimique , Agatoxines , Protéine de signalisation Agouti , Protéine apparentée à Agouti , Alkylation , Séquence d'acides aminés , Animaux , Cystéine/composition chimique , Disulfures/métabolisme , Fluorescéines/métabolisme , Humains , Données de séquences moléculaires , Oxydoréduction , Fragments peptidiques/composition chimique , Fragments peptidiques/isolement et purification , Fragments peptidiques/métabolisme , Protéines/isolement et purification , Protéines/métabolisme , Venins d'araignée/composition chimiqueRÉSUMÉ
Gene targeting has proven to be extremely powerful in various fields of biological research. Through this technique, knockout mice lacking a particular gene, and thus a particular protein, can be generated. One limitation to this technique is the fact that mice develop without the protein of interest and therefore, developmental compensations may have taken place, contributing to an observed phenotype. Inducible strategies, those which allow the timing of expression of a gene to be regulated, are currently being developed and should prove useful when applied to gene targeting technology. To begin to apply such new technologies to the field of gene targeting, we first created and tested several reporter constructions using the tetracycline inducible system. Here we describe the creation of several beta-galactosidase reporter constructions and the results of in vitro testing in Cos-7 cells. We then discuss future knockout strategies based upon our observations.
Sujet(s)
Ciblage de gène , Récepteurs sérotoninergiques/physiologie , Animaux , Encéphale/physiologie , Cellules COS , Régulation de l'expression des gènes/physiologie , Gènes rapporteurs/génétique , Humains , Souris , Souris knockout , Récepteurs sérotoninergiques/génétique , beta-Galactosidase/génétiqueRÉSUMÉ
We have developed a new procedure utilizing microhomologous recombination in yeast to generate targeting constructs for producing targeted mutations in mice. This procedure is rapid and efficient, and should be directly applicable to all mammalian genes. Moreover, only minimal information about the locus being targeted is required. The feasibility of this approach was demonstrated by producing another allele of the mouse Tg737 polycystic kidney gene.
Sujet(s)
Ciblage de gène , Mutagenèse , Polykystoses rénales/génétique , Recombinaison génétique , Saccharomyces cerevisiae/génétique , Allèles , Animaux , Séquence nucléotidique , Chimère , Clonage moléculaire/méthodes , Amorces ADN , Exons , Vecteurs génétiques , Banque génomique , Mammifères , Souris , Lignées consanguines de souris , Données de séquences moléculaires , Réaction de polymérisation en chaîne , Transformation génétiqueRÉSUMÉ
Rapid progress has recently been made to characterise a new pathway involved in the control of feeding behaviour, referred to as the melanocortinergic pathway. Studies of the obese phenotype of the yellow mouse mutant (Ay) suggested the existence of this feeding circuit for some time, but the precise molecular and biochemical regulation was unclear. Through pharmacological and genetic approaches, specific melanocortin receptors expressed within the brain are now known to play a pivotal role. Since receptor agonists decrease food intake, and blocking receptor activity (through antagonists or mutations) increases food intake, this pathway is thought to provide a tonic inhibitory effect on food consumption. A unique feature of this pathway seems to be the regulation of receptor activity by an endogenous protein antagonist, agouti-related protein (AGRP). The molecular characterisation and biochemical activity of this gene suggest that it plays a role in the regulation of feeding behaviour.
RÉSUMÉ
Gene targeting has proven to be extremely powerful in various fields of biological research. Through this technique, knockout mice lacking a particular gene and thus a particular protein, can be generated. One limitation to this technique is the fact that mice develop without the protein of interest and therefore, developmental compensations may have taken place, contributing to an observed phenotype. Inducible strategies, those which allow the timing of expression of a gene to be regulated, are currently being developed and should prove useful when applied to gene targeting technology. In order to begin to apply such new technologies to the field of gene targeting, we first created and tested several reporter constructions using the tetracycline inducible system. Here we describe the creation of several beta-galactosidase reporter constructions and the results of in vitro testing in Cos-7 cells. We then discuss future knockout strategies based upon our observations.
Sujet(s)
Récepteurs sérotoninergiques/physiologie , Animaux , Cellules COS , Régulation de l'expression des gènes , Gènes rapporteurs , Kanamycin kinase/biosynthèse , Kanamycin kinase/génétique , Souris , Souris knockout , Mutagenèse par insertion , Plasmides , Récepteurs sérotoninergiques/déficit , Récepteurs sérotoninergiques/génétique , Protéines recombinantes/biosynthèse , Transfection/méthodes , beta-Galactosidase/biosynthèse , beta-Galactosidase/génétiqueSujet(s)
Souris obèse/génétique , Protéines/génétique , Actines/génétique , Tissu adipeux/anatomopathologie , Protéine apparentée à Agouti , Animaux , Poids/génétique , Corticostérone/sang , Modèles animaux de maladie humaine , Femelle , Insuline/sang , Protéines et peptides de signalisation intercellulaire , Foie/anatomopathologie , Mâle , Souris , Souris transgéniques , Obésité/génétique , Protéines/métabolisme , Transgènes , Prise de poids/génétiqueRÉSUMÉ
The mCAT-1 gene encodes a basic amino acid transporter that also acts as the receptor for murine ecotropic leukemia viruses. Targeted mutagenesis in embryonic stem cells has been used to introduce a germ-line null mutation into this gene. This mutation removes a domain critical for virus binding and inactivates amino acid transport activity. Homozygous mutant pups generated from these cells were approximately 25% smaller than normal littermates, very anemic, and died on the day of birth. Peripheral blood from homozygotes contained 50% fewer red blood cells, reduced hemoglobin levels, and showed a pronounced normoblastosis. Histological analyses of bone marrow, spleen, and liver showed a decrease in both erythroid progenitors and mature red blood cells. Mutant fetal liver cells behaved normally in in vitro hematopoietic colony-forming assays but generated an anemia when transplanted into irradiated C.B.-17 SCID mice. Furthermore, reconstitution of the white cell compartment of SCID mice by mutant fetal liver cells was less complete than that observed with a mixed population of wild-type and heterozygous fetal liver cells. Primary embryo fibroblasts from mutant mice were completely resistant to ecotropic retrovirus infection. Thus, mCAT-1 not only appears to be the sole receptor for a group of murine ecotropic retroviruses associated with hematological disease but also plays a critical role in both hematopoiesis and growth control during mouse development.
Sujet(s)
Anémie/congénital , Protéines de transport/génétique , Gènes létaux , Glycoprotéines membranaires , Protéines membranaires/génétique , Récepteurs viraux/génétique , Retroviridae/croissance et développement , Animaux , Animaux nouveau-nés , Moelle osseuse/malformations , Numération cellulaire , Transplantation cellulaire , Précurseurs érythroïdes/cytologie , Hématopoïèse/génétique , Foie/malformations , Transplantation hépatique , Souris , Souris knockout , Souris SCID , Rate/malformations , Cellules souchesRÉSUMÉ
We have isolated cDNA clones that encode a novel human gene related to agouti. Sequence analysis of this gene, named ART, for agouti-related transcript, predicts a 132-amino-acid protein that is 25% identical to human agouti. The highest degree of identity is within the carboxyl terminus of both proteins. Like agouti, ART contains a putative signal sequence and a cysteine rich carboxyl terminus, but lacks the region of basic residues and polyproline residues found in the middle of the agouti protein. Both agouti and ART contain 11 cysteines, and 9 of these are conserved spatially. ART is expressed primarily in the adrenal gland, subthalamic nucleus, and hypothalamus, with a lower level of expression occurring in testis, lung, and kidney. The murine homolog of ART was also isolated and is predicted to encode a 131-amino-acid protein that shares 81% amino acid identity to humans. The mouse was found to have the same expression pattern as human when assessed by RT-PCR. Examination by in situ hybridization using mouse tissues showed localized expression in the arcuate nucleus of the hypothalamus, the median eminence, and the adrenal medulla. In addition, the hypothalamic expression of ART was elevated approximately 10-fold in ob/ob and db/db mice. ART was mapped to human chromosome 16q22 and to mouse chromosome 8D1-D2. The expression pattern and transcriptional regulation of ART, coupled with the known actions of agouti, suggests a role for ART in the regulation of melanocortin receptors within the hypothalamus and adrenal gland, and implicates this novel gene in the central control of feeding.
Sujet(s)
Hypothalamus/métabolisme , Protéines et peptides de signalisation intercellulaire , Souris obèse/génétique , Protéines/génétique , Protéines/métabolisme , Protéine de signalisation Agouti , Protéine apparentée à Agouti , Épissage alternatif , Séquence d'acides aminés , Animaux , Séquence nucléotidique , Technique de Northern , Calcium/métabolisme , Cartographie chromosomique , Chromosomes humains de la paire 16 , Clonage moléculaire , Séquence conservée , Bases de données factuelles , Diabète expérimental/génétique , Modèles animaux de maladie humaine , Humains , Souris , Souches mutantes de souris , Données de séquences moléculaires , Famille multigénique , Mutation , Obésité/génétique , Protéines/composition chimique , Récepteur corticotrophine/métabolisme , Récepteurs à la mélanocortine , Alignement de séquences , Similitude de séquences d'acides aminés , Transduction du signal , Distribution tissulaire , Transcription génétique , Régulation positiveRÉSUMÉ
Amphibian studies have implicated Wnt signaling in the regulation of mesoderm formation, although direct evidence is lacking. We have characterized the expression of 12 mammalian Wnt-genes, identifying three that are expressed during gastrulation. Only one of these, Wnt-3a, is expressed extensively in cells fated to give rise to embryonic mesoderm, at egg cylinder stages. A likely null allele of Wnt-3a was generated by gene targeting. All Wnt-3a-/Wnt-3a- embryos lack caudal somites, have a disrupted notochord, and fail to form a tailbud. Thus, Wnt-3a may regulate dorsal (somitic) mesoderm fate and is required, by late primitive steak stages, for generation of all new embryonic mesoderm. Wnt-3a is also expressed in the dorsal CNS. Mutant embryos show CNS dysmorphology and ectopic expression of a dorsal CNS marker. We suggest that dysmorphology is secondary to the mesodermal and axial defects and that dorsal patterning of the CNS may be regulated by inductive signals arising from surface ectoderm.
Sujet(s)
Développement embryonnaire et foetal/génétique , Protéines/génétique , Animaux , Séquence nucléotidique , Système nerveux central/embryologie , Gastrula , Mésoderme , Souris , Données de séquences moléculaires , Oligodésoxyribonucléotides , Queue/embryologie , Protéines de type Wingless , Protéine Wnt3 , Protéine Wnt3ARÉSUMÉ
We have used the polymerase chain reaction to clone from fetal cerebellar RNA a novel member of the fibroblast growth factor receptor family, FGFR-4. cDNAs encoding a full-length receptor were isolated and RNA expression examined in adult and fetal tissues by RNA blot analysis. Transcripts were detected in adult lung, liver and kidney and in fetal RNAs from 11.5 to 16.5 days post coitum (p.c.). In situ hybridization was performed to examine developmental expression. FGFR-4 RNA was expressed in definitive endoderm of the developing gut and extraembryonic endoderm of the yolk-sac from 8.5 to 14.5 days p.c. At early somite stages, FGFR-4 was also expressed in the myotomal component of the somite, and by 14.5 days p.c. in the myotomally derived skeletal muscle. No expression was seen at any stage in cardiac muscle. Several endodermal derivatives, the liver, lung and pancreas, expressed FGFR-4 at 14.5 days p.c. In addition, FGFR-4 RNA was detected in the adrenal cortex, collecting tubules of the kidney and condensing cartilage at this time. These results suggest that FGFR-4 is likely to have diverse roles in development, which may include regulation of definitive endoderm and skeletal muscle lineages.
Sujet(s)
Endoderme/physiologie , Facteurs de croissance fibroblastique/physiologie , Muscles/physiologie , Récepteurs de surface cellulaire/génétique , Récepteur facteur croissance fibroblaste , Séquence d'acides aminés , Animaux , Séquence nucléotidique , Clonage moléculaire , Endoderme/composition chimique , Souris , Techniques de sonde moléculaire , Données de séquences moléculaires , Réaction de polymérisation en chaîne , ARN/analyse , Récepteur FGFR4RÉSUMÉ
These investigations were undertaken to determine the extent to which tissues of cultured rat conceptuses contain cytochrome P450 isoforms in sufficient quantities to significantly influence the capacity of certain chemicals to elicit dysmorphogenic effects in vitro. Investigations with highly sensitive probe substrates/inhibitors and with immunologic methods enabled the detection of at least four separate P450 isoforms in tissues of the visceral yolk sac, ectoplacental cone, and embryo proper. One of the isoforms was identified as P450IA1 and was found to be inducible by polycyclic aromatic hydrocarbons in all three tissues. Other isoforms exhibited properties differing from characterized adult rat hepatic isoforms. Each of the isoforms was detectable in conceptuses on gestational days 10, 11, 12, and 14 and was present in the highest concentrations in the visceral yolk sac. Conceptal P450IA1 catalyzed the conversion of dysmorphogenically inactive 2-acetylaminofluorene to 7-hydroxy-2-acetylaminofluorene, a proximate dysmorphogen. Investigations with microinjections suggested that visceral yolk sac hydroxylation was largely responsible for the bioactivation reaction in vitro. The same isoform exhibited no capacity to influence the dysmorphogenic activity of cyclophosphamide. The results demonstrated that tissues of cultured rat conceptuses may contain P450 isoforms in sufficient amounts to markedly influence the dysmorphogenic activity of substrates of the corresponding isoforms.