RESUMO
Development of the vertebrate limb bud depends on reciprocal interactions between the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER). Sonic hedgehog (SHH) and fibroblast growth factors (FGFs) are key signalling molecules produced in the ZPA and AER, respectively. Experiments in chicks suggested that SHH expression in the ZPA is maintained by FGF4 expression in the AER, and vice versa, providing a molecular mechanism for coordinating the activities of these two signalling centres. This SHH/FGF4 feedback loop model is supported by genetic evidence showing that Fgf4 expression is not maintained in Shh-/- mouse limbs. We report here that Shh expression is maintained and limb formation is normal when Fgf4 is inactivated in mouse limbs, thus contradicting the model. We also found that maintenance of Fgf9 and Fgf17 expression is dependent on Shh, whereas Fgf8 expression is not. We discuss a model in which no individual Fgf expressed in the AER (AER-Fgf) is solely necessary to maintain Shh expression, but, instead, the combined activities of two or more AER-Fgfs function in a positive feedback loop with Shh to control limb development.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Botões de Extremidades/embriologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Superfície Celular , Transdução de Sinais/genética , Transativadores , Proteínas Virais , Animais , Proteínas de Ligação a DNA/genética , Ectoderma/metabolismo , Ectoderma/fisiologia , Proteínas do Ovo/genética , Retroalimentação/fisiologia , Fator 4 de Crescimento de Fibroblastos , Genes Letais , Proteínas Hedgehog , Proteínas de Homeodomínio , Integrases/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas/genética , Zona Pelúcida/fisiologia , Glicoproteínas da Zona PelúcidaRESUMO
The genetic analysis of congenital skull malformations provides insight into normal mechanisms of calvarial osteogenesis. Enlarged parietal foramina (PFM) are oval defects of the parietal bones caused by deficient ossification around the parietal notch, which is normally obliterated during the fifth fetal month. PFM are usually asymptomatic, but may be associated with headache, scalp defects and structural or vascular malformations of the brain. Inheritance is frequently autosomal dominant, but no causative mutations have been identified in non-syndromic cases. We describe here heterozygous mutations of the homeobox gene MSX2 (located on 5q34-q35) in three unrelated families with PFM. One is a deletion of approximately 206 kb including the entire gene and the others are intragenic mutations of the DNA-binding homeodomain (RK159-160del and R172H) that predict disruption of critical intramolecular and DNA contacts. Mouse Msx2 protein with either of the homeodomain mutations exhibited more than 85% reduction in binding to an optimal Msx2 DNA-binding site. Our findings contrast with the only described MSX2 homeodomain mutation (P148H), associated with craniosynostosis, that binds with enhanced affinity to the same target. This demonstrates that MSX2 dosage is critical for human skull development and suggests that PFM and craniosynostosis result, respectively, from loss and gain of activity in an MSX2-mediated pathway of calvarial osteogenic differentiation.
Assuntos
Suturas Cranianas/anormalidades , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Mutação , Crânio/anormalidades , Adulto , Animais , Sequência de Bases , Southern Blotting , Criança , Pré-Escolar , Cromossomos Humanos Par 5/genética , Suturas Cranianas/diagnóstico por imagem , Suturas Cranianas/crescimento & desenvolvimento , Análise Mutacional de DNA , Proteínas de Ligação a DNA/deficiência , Feminino , Humanos , Lactente , Masculino , Camundongos , Repetições de Microssatélites , Dados de Sequência Molecular , Osteogênese/genética , Osso Parietal/anormalidades , Osso Parietal/crescimento & desenvolvimento , Linhagem , Radiografia , Deleção de Sequência , Crânio/diagnóstico por imagem , Crânio/crescimento & desenvolvimentoRESUMO
Msx2 is a homeobox gene expressed in multiple embryonic tissues which functions as a key mediator of numerous developmental processes. YY1 is a bi-functional zinc finger protein that serves as a repressor or activator to a variety of promoters. The role of YY1 during embryogenesis remains unknown. In this study, we report that Msx2 is regulated by YY1 through protein-DNA interactions. During embryogenesis, the expression pattern of YY1 was observed to overlap in part with that of Msx2. Most notably, during first branchial arch and limb development, both YY1 and Msx2 were highly expressed, and their patterns were complementary. To test the hypothesis that YY1 regulates Msx2 gene expression, P19 embryonal cells were used in a number of expression and binding assays. We discovered that, in these cells, YY1 activated endogenous Msx2 gene expression as well as Msx2 promoter-luciferase fusion gene activity. These biological activities were dependent on both the DNA binding and activation domains of YY1. In addition, YY1 bound specifically to three YY1 binding sites on the proximal promoter of Msx2 that accounted for this transactivation. Mutations introduced to these sites reduced the level of YY1 transactivation. As bone morphogenetic protein type 4 (BMP4) regulates Msx2 expression in embryonic tissues and in P19 cells, we further tested whether YY1 is the mediator of this BMP4 activity. BMP4 did not induce the expression of YY1 in early mouse mandibular explants, nor in P19 cells, suggesting that YY1 is not a required mediator of the BMP4 pathway in these tissues at this developmental stage. Taken together, these findings suggest that YY1 functions as an activator for the Msx2 gene, and that this regulation, which is independent of the BMP4 pathway, may be required during early mouse craniofacial and limb morphogenesis.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/fisiologia , Ativação Transcricional , Animais , Proteína Morfogenética Óssea 4 , Região Branquial/embriologia , Região Branquial/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Ligação de DNA Eritroide Específicos , Extremidades/embriologia , Genes Reporter , Proteínas de Homeodomínio , Hibridização In Situ , Mandíbula/efeitos dos fármacos , Mandíbula/embriologia , Mandíbula/metabolismo , Camundongos , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA Mensageiro/biossíntese , Transdução de Sinais , Transativadores/química , Transativadores/genética , Transativadores/fisiologia , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transfecção , Células Tumorais Cultivadas , Fator de Transcrição YY1RESUMO
To facilitate the elucidation of the genetic events that may play an important role in the development or tumorigenesis of the prostate gland, we have generated a transgenic mouse line with prostate-specific expression of Cre recombinase. This line, named PB-Cre4, carries the Cre gene under the control of a composite promoter, ARR2PB which is a derivative of the rat prostate-specific probasin (PB) promoter. Based on RT-PCR detection of Cre mRNA in PB-Cre4 mice or Cre-mediated activation of LacZ activity in PB-Cre4/R26R double transgenic mice, it is conclusively demonstrated that Cre expression is post-natal and prostatic epithelium-specific. Although the Cre recombination is detected in all lobes of the mouse prostate, there is a significant difference in expression levels between the lobes, being highest in the lateral lobe, followed by the ventral, and then the dorsal and anterior lobes. Besides the prostate gland, no other tissues of the adult PB-Cre4 mice demonstrate significant Cre expression, except for a few scattered areas in the gonads and the stroma of the seminal vesicle. By crossing the PB-Cre4 animals with floxed RXRalpha allelic mice, we demonstrate that mice, whose conventional knockout of this gene is lethal in embryogenesis, could be propagated with selective inactivation of RXRalpha in the prostate. Taken together, the results show that the PB-Cre4 mice have high levels of Cre expression and a high penetrance in the prostatic epithelium. The PB-Cre4 mice will be a useful resource for genetic-based studies on prostate development and prostatic disease.
Assuntos
Epitélio/metabolismo , Integrases/biossíntese , Integrases/genética , Próstata/metabolismo , Proteínas Virais , Alelos , Animais , Cruzamentos Genéticos , Feminino , Galactosídeos/metabolismo , Imuno-Histoquímica , Indóis/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Camundongos Transgênicos , Ovário/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Próstata/crescimento & desenvolvimento , Neoplasias da Próstata/metabolismo , Ratos , Receptores do Ácido Retinoico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/metabolismo , Fatores de Tempo , Distribuição Tecidual , TransgenesRESUMO
The homeobox gene Msx-2 is expressed specifically in sites of skin appendage formation. To explore its part in skin morphogenesis, we produced transgenic mice expressing Msx-2 under the control of the cytomegalovirus promoter. The skin of these transgenic mice was flaky, exhibiting desquamation and shorter hairs. Histologic analysis showed thickened epidermis with hyperproliferation, which was restricted to the basal layer. Hyperkeratosis was also evident. A wide zone of suprabasal cells were misaligned and coexpressed keratins 14 and 10. There was reduced expression of integrin beta 1 and DCC in the basal layer. Hair follicles were misaligned with a shrunken matrix region. The dermis showed increased cellularity and empty vacuoles. We suggest that Msx-2 is involved in the growth control of skin and skin appendages.
Assuntos
Anormalidades Múltiplas/genética , Proteínas de Ligação a DNA/genética , Epiderme/anormalidades , Genes Homeobox/genética , Folículo Piloso/anormalidades , Animais , Expressão Gênica , Proteínas de Homeodomínio , Camundongos , Camundongos Transgênicos , Pele/crescimento & desenvolvimentoRESUMO
Catecholamines were determined by a radioenzymatic assay in maternal and fetal rat plasma and in the amniotic fluid during the last five days of gestation. Dopamine was significantly higher in fetal than in maternal plasma whereas norepinephrine was the same. Epinephrine was sighificantly lower in fetal than in maternal plasma on days 18 and 19, but was the same during the remainder of gestation. Dopamine was the predominant catecholamine in the amniotic fluid on days 20-22 of gestation showing the largest percent increase prior to parturition. The data suggest that dopamine may have a peripheral hormonal role during fetal development and parturition.
Assuntos
Líquido Amniótico/metabolismo , Dopamina/metabolismo , Sangue Fetal/metabolismo , Animais , Epinefrina/metabolismo , Feminino , Idade Gestacional , Norepinefrina/metabolismo , Gravidez , RatosRESUMO
Nucleotide (nt) sequence analyses of the 1.2-kb BamHI-EcoRI cloned 3'-fragment encompassing the polymorphic SmaI restriction site of the feline c-myc gene reveal that the SmaI site, present in CM2 allele but absent from CM3 allele, is located in intron 2, 134 nt 5' of the exon 3. A G-to-C transversion in CM2 results in the creation of the SmaI site. Additionally, the alleles differ at four other nt positions in intron 2, three of these changes being in a region of the intron which exhibits 80% homology between the feline and human c-myc. The alleles also differ in two nt positions in exon 3 in the third position of the codon resulting, however, in no amino acid alteration. Genotype distribution analysis based on the SmaI polymorphism shows that CM2 homozygosity is rare and its frequency deviates significantly from the expected distribution patterns for independently segregating alleles.
Assuntos
Gatos/genética , Proto-Oncogenes , Alelos , Animais , Sequência de Bases , Doenças do Gato/genética , Frequência do Gene , Genótipo , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/veterináriaRESUMO
Many genetically determined craniosynostosis syndromes feature limb anomalies, implying that pathways of cranial suture and limb morphogenesis share some identical components. Identification of heterozygous mutations in FGFR1, FGFR2, FGFR3, TWIST and MSX2 in craniosynostosis has focused particular attention on these genes. Here we explore two themes: use of clinical/molecular analysis to provide new clues to pathophysiology and the contrasting effects of loss- and gain-of-function mutations. Apert syndrome is a severe craniosynostosis/syndactyly disorder usually caused by specific substitutions (Ser252Trp or Pro253Arg) in FGFR2. The relative severity of cranial and limb malformations varies in opposite directions for the two mutations, suggesting that these phenotypes arise by different mechanisms. Clinical and biochemical evidence supports a model in which alternative splice forms of FGFR2 mediate these distinct effects. Pro-->Arg substitutions equivalent the Pro253Arg/FGFR2 mutation occur in both FGFR1 and FGFR3, and are also associated with craniosynostosis. This suggests a common pathological mechanism, whereby enhanced affinity for a limited repertoire of tissue-specific ligand(s) excessively prolongs signalling in the cranial suture. The first MSX2 mutation in craniosynostosis was described in 1993 but this remains the only example. We have recently identified three MSX2 mutations associated with a different cranial phenotype, parietal foramina. DNA binding studies show that the craniosynostosis and parietal foramina arise from gain and loss of function, respectively.
Assuntos
Acrocefalossindactilia/genética , Braço/anormalidades , Craniossinostoses/genética , Perna (Membro)/anormalidades , Mutação , Proteínas Nucleares , Proteínas Tirosina Quinases , Receptores de Fatores de Crescimento de Fibroblastos/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Sequências Hélice-Alça-Hélice , Proteínas de Homeodomínio/genética , Humanos , Dados de Sequência Molecular , Morfogênese , Estrutura Secundária de Proteína , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/química , Fatores de Transcrição/genética , Proteína 1 Relacionada a TwistAssuntos
Anormalidades Craniofaciais/genética , Craniossinostoses/genética , Proteínas de Ligação a DNA/genética , Fosfatase Alcalina/genética , Animais , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Genes Letais , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Transgênicos , Fenótipo , Pró-Colágeno/genética , Regiões Promotoras GenéticasRESUMO
We describe a method for the rapid, one-step determination of the specific radioactivity and pool size of ATP, UTP, CTP or GTP. Escherichia coli RNA polymerase and poly[d(A-T)] or poly[d(G-C)] are used to synthesize an alternating copolymer from a [3H]nucleoside triphosphate of unknown specific activity and a [14C]nucleoside triphosphate of known specific activity. The fact that [3H]nucleotide and [14C]nucleotide are incorporated into poly[r(A-U)] or poly[r(G-C)] in equimolar amounts, coupled with a knowledge of the [14C]nucleotide specific activity, permits calculation of the [3H]nucleotide specific activity. The requirement for direct knowledge of the [14C]nucleotide specific activity may be bypassed by an isotope dilution procedure. The pool size of a nucleoside triphosphate can be estimated either from isotope dilution data or by determining the fraction of [3H]nucleotide polymerized, dividing the number of counts 3H/min in the polymer by this fraction and by the [3H]nucleotide specific activity. The method was successfully applied to acid extracts made from sea urchin embryos labeled with a [3H]RNA precursor.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Ribonucleotídeos/análise , Animais , Radioisótopos de Carbono , Escherichia coli/enzimologia , Feminino , Marcação por Isótopo , Cinética , Matemática , Métodos , Óvulo/análise , Ouriços-do-Mar/análise , Especificidade da Espécie , TrítioRESUMO
A fast transport of noradrenaline (NA) at a velocity of 392 mm/day was found in cat peroneal nerve using a double-ligation technique and a new sensitive radioenzymatic assay for catecholamines. The velocity of transport of NA is sufficiently close to that of 410 mm/day found for labelled proteins and polypeptides to be considered as moving down within the nerve fibres by the same transport mechanism. In addition, dopamine (DA) was also found to be moved down by anterograde transport at a fast, but not well defined, rate. Disulfiram, a dopamine-beta-hydroxylase inhibiting agent, reduced NA levels and increased DA both in control nerve segments and within the portion of the nerve isolated by ligations where NA-containing densecore vesicles are present. The results are considered to support a dynamic turnover of NA and DA in the dense-core vesicles as they are trasnported in the axons.
Assuntos
Transporte Axonal , Dopamina/fisiologia , Norepinefrina/fisiologia , Nervos Periféricos/fisiologia , Animais , Transporte Axonal/efeitos dos fármacos , Axônios/fisiologia , Gatos , Dissulfiram/farmacologiaRESUMO
In this review, we provide a survey of the experimental approaches used to generate genetically engineered mice. Two specific examples are presented that demonstrate the applicability of these approaches to craniofacial development. In the first, a promoter analysis of the Msx2 gene is presented which illustrates the cis regulatory interactions that defined cell-specific gene expression. In the second, a mouse model of the human disease craniosynostosis, Boston type, has been created by misregulation of the Msx2 gene product. Finally. we present a formulary of spontaneously occurring and genetically engineered mice that exhibit defects in developmental processes affecting the craniofacial complex. The purpose of this review is to provide insight into the experimental approaches that are used to create genetically engineered mice and to impress upon the reader that genetically engineered mice are well-suited to address fundamental questions pertaining to the development maintenance, and regeneration of tissues and organs.
Assuntos
Engenharia Genética , Desenvolvimento Maxilofacial/genética , Camundongos Transgênicos , Animais , Craniossinostoses/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox/genética , Genes Reguladores/genética , Proteínas de Homeodomínio/genética , Camundongos , Regiões Promotoras Genéticas/genética , Regeneração/genéticaRESUMO
The coordinate growth of the brain and skull is achieved through a series of interactions between the developing brain, the growing bones of the skull, and the fibrous joints, or sutures, that unite the bones. These interactions couple the expansion of the brain to the growth of the bony plates at the sutures. Craniosynostosis, the premature fusion of the bones of the skull, is a common birth defect (1 in 3000 live births) that disrupts coordinate growth and often results in profoundly abnormal skull shape. Individuals affected with Boston-type craniosynostosis, an autosomal dominant disorder, bear a mutated copy of MSX2, a homeobox gene thought to function in tissue interactions. Here we show that expression of the mouse counterpart of this mutant gene in the developing skulls of transgenic mice causes craniosynostosis and ectopic cranial bone. These mice provide a transgenic model of craniosynostosis as well as a point of entry into the molecular mechanisms that coordinate the growth of the brain and skull.
Assuntos
Craniossinostoses/genética , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Crânio/anormalidades , Crânio/embriologia , Animais , Craniossinostoses/embriologia , Proteínas de Ligação a DNA/biossíntese , Feminino , Genes Homeobox , Histidina , Humanos , Hibridização In Situ , Recém-Nascido , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Mutação Puntual , Prolina , Crânio/citologia , Crânio/metabolismoRESUMO
The Msx homeobox genes are expressed in complex patterns during vertebrate development in conjunction with inductive tissue interactions. As a means of understanding the archetypal role of Msx genes in chordates, we have isolated and characterized an Msx gene in ascidians, protochordates with a relatively simple body plan. The Mocu Msx-a and McMsx-a genes, isolated from the ascidians Molgula oculata and Molgula citrina, respectively, have homeodomains that place them in the msh-like subclass of Msx genes. Therefore, the Molgula Msx-a genes are most closely related to the msh genes previously identified in a number of invertebrates. Southern blot analysis suggests that there are one or two copies of the Msx-a gene in the Molgula genome. Northern blot and RNase protection analysis indicate that Msx-a transcripts are restricted to the developmental stages of the life cycle. In situ hybridization showed that Msx-a mRNA first appears just before gastrulation in the mesoderm (presumptive notochord and muscle) and ectoderm (neural plate) cells. Transcript levels decline in mesoderm cells after the completion of gastrulation, but are enhanced in the folding neural plate during neurulation. Later, Msx-a mRNA is also expressed in the posterior ectoderm and in a subset of the tail muscle cells. The ectoderm and mesoderm cells that express Msx-a are undergoing morphogenetic movements during gastrulation, neurulation, and tail formation. Msx-a expression ceases after these cells stop migrating. The ascidian M. citrina, in which adult tissues and organs begin to develop precociously in the larva, was used to study Msx-a expression during adult development. Msx-a transcripts are expressed in the heart primordium and the rudiments of the ampullae, epidermal protrusions with diverse functions in the juvenile. The heart and ampullae develop in regions where mesenchyme cells interact with endodermal or epidermal epithelia. A comparison of the expression patterns of the Molgula genes with those of their vertebrate congeners suggests that the archetypal roles of the Msx genes may be in morphogenetic movements during embryogenesis and in mesenchymal-epithelial interactions during organogenesis.
Assuntos
Expressão Gênica , Genes Homeobox , Urocordados/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA , Dados de Sequência Molecular , Filogenia , RNA Mensageiro , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
We are interested in understanding hierarchical regulation pathways that control gene expression in developing teeth. In pursuit of the molecular basis for the regulated expression of amelogenin by developing ameloblasts during tooth formation, we isolated the murine amelogenin promoter. Analysis of this promoter will provide additional details towards the identification of signals generated through instructive-, dissimilar-germ layer interactions that are for responsible for temporal- and spatial-regulation for amelogenin gene expression. Using transgenic mice we demonstrate that a 2263 nucleotide stretch of the murine amelogenin promoter conveys appropriate temporal- and spatial-regulation for amelogenin gene expression in response to instructive-signals. These transgenic animals are useful reagents to further dissect signaling pathways responsible for regulated gene expression by terminally differentiated ameloblasts.
Assuntos
Ameloblastos/metabolismo , Proteínas do Esmalte Dentário/genética , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas , Células 3T3 , Amelogenina , Animais , Animais Geneticamente Modificados , Feminino , Genes Reporter , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Dente Molar/metabolismoRESUMO
Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998) Development 125, 1241-1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6). Twist and Id1 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated that in Twist(+/)(-) mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disruption of this pathway may lead to craniosynostosis.
Assuntos
Fator 2 de Crescimento de Fibroblastos/genética , Proteínas Nucleares/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Proteínas Repressoras , Crânio/metabolismo , Fator de Crescimento Transformador beta , Acrocefalossindactilia/genética , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular , Craniossinostoses/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Sequências Hélice-Alça-Hélice , Humanos , Imuno-Histoquímica , Hibridização In Situ , Proteína 1 Inibidora de Diferenciação , Sialoproteína de Ligação à Integrina , Camundongos , Proteínas Nucleares/metabolismo , Osteogênese/genética , Sialoglicoproteínas/genética , Transdução de Sinais/genética , Crânio/embriologia , Crânio/crescimento & desenvolvimento , Fatores de Transcrição/genética , Proteína 1 Relacionada a TwistRESUMO
Throughout its complex morphogenesis, the vertebrate skull must at once protect the brain and expand to accommodate its growth. A key structural adaptation that allows this dual role is the separation of the bony plates of the skull with sutures, fibrous joints that serve as growth centers and allow the calvarial bones to expand as the brain enlarges. Craniosynostosis, the premature fusion of one or more calvarial bones with consequent abnormalities in skull shape, is a common developmental anomaly that disrupts this process. We found previously that a single amino acid substitution in the homeodomain of the human MSX2 gene is associated with the autosomal dominant disorder craniosynostosis, Boston type. This mutation enhances the affinity of Msx2 for its target sequence, suggesting that the mutation acts by a dominant positive mechanism. Consistent with this prediction, we showed that general overexpression of Msx2 under the control of the broadly expressed CMV promoter causes the calvarial bones to invade the sagittal suture. Here we use tissue-specific overexpression of Msx2 within the calvarial sutures to address the developmental mechanisms of craniosynostosis and skull morphogenesis. We demonstrate that a segment of the Msx2 promoter directs reporter gene expression to subsets of cells within the sutures. In late embryonic and neonatal stages, this promoter is expressed in undifferentiated mesenchymal cells medial to the growing bone. By P4, promoter activity is reduced in the suture, exhibiting a punctate pattern in undifferentiated osteoblastic cells in the outer margin of the osteogenic front. Overexpression of Msx2 under the control of this promoter is sufficient to enhance parietal bone growth into the sagittal suture by P6. This phenotype is preceded by an increase in both the number and the BrdU labeling of osteoblastic cells in the osteogenic fronts of the calvarial bones. These findings suggest that an important early event in MSX2-mediated craniosynostosis in humans is a transient retardation of osteogenic cell differentiation in the suture and a consequent increase in the pool of osteogenic cells.