RESUMO
In situ hybridization to cellular RNA is used to determine the cellular localization of specific messages within complex cell populations and tissues. In this unit, protocols are described for hybridizing slide-mounted paraffin sections or cryosections with labeled probes. Support protocols describe synthesis of 35S-labeled riboprobes and dsDNA probes, which are then detected using film autoradiography or emulsion autoradiography. Another support protocol describes synthesis of digoxigenin-labeled RNA probes, which are non-radioactive and thus have several advantages. They are easily synthesized in large quantities, they are stable for several months, and they can be reused up to three times. An additional advantage of RNA versus DNA probes is that they result in cleaner signals because nonspecifically bound probe is removed during ribonuclease treatment.
Assuntos
Hibridização In Situ/métodos , RNA/genética , Digoxigenina , Indicadores e Reagentes , Desnaturação de Ácido Nucleico , Sondas RNA , RNA Mensageiro/genética , Transcrição GênicaRESUMO
In situ hybridization to cellular RNA is used to determine the cellular localization of specific messages within complex cell populations and tissues. Tissues may either be embedded in paraffin and sectioned on a microtome (see Hybridization Using Paraffin Sections and Cells), or frozen and sectioned in a cryostat (see Hybridization Using Cryosections). RNA contained in the specimens is hybridized to a specific radiolabeled probe (see Synthesis of (35)S-Labled Riboprobes and Synthesis of (35)S-Labled Double-Stranded DNA Probes), which is then detected using film autoradiography or emulsion autoradiography (Chapter 8).
Assuntos
Hibridização In Situ/métodos , RNA/análise , Autorradiografia/métodos , Crioultramicrotomia/métodos , Sondas de DNA/metabolismo , Digoxigenina , Indicadores e Reagentes , Inclusão em Parafina/métodos , Sondas RNA/metabolismo , Soluções , Manejo de Espécimes/métodos , Radioisótopos de Enxofre , Fixação de Tecidos/métodosRESUMO
Formin was originally isolated as the gene affected by the murine limb deformity (ld) mutations, which disrupt the epithelial-mesenchymal interactions regulating patterning of the vertebrate limb autopod. More recently, a rapidly growing number of genes with similarity to formin have been isolated from many different species including fungi and plants. Genetic and biochemical analysis shows that formin family members function in cellular processes regulating either cytokinesis and/or cell polarisation. Another common feature among formin family members is their requirement in morphogenetic processes such as budding and conjugation of yeast, establishment of Drosophila oocyte polarity and vertebrate limb pattern formation. Vertebrate formins are predominantly nuclear proteins which control polarising activity in limb buds through establishment of the SHH/FGF-4 feedback loop. Formin acts in the limb bud mesenchyme to induce apical ectodermal ridge (AER) differentiation and FGF-4 expression in the posterior AER compartment. Finally, disruption of the epithelial-mesenchymal interactions controlling induction of metanephric kidneys in ld mutant embryos indicates that formin might function more generally in transduction of morphogenetic signals during embryonic pattern formation.
Assuntos
Padronização Corporal/genética , Extremidades/embriologia , Proteínas Fetais/genética , Deformidades Congênitas dos Membros/genética , Morfogênese/genética , Família Multigênica , Proteínas Nucleares/genética , Sequência de Aminoácidos , Animais , Evolução Molecular , Proteínas Fetais/química , Proteínas Fetais/fisiologia , Forminas , Humanos , Camundongos , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/fisiologia , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
Mutations in the murine limb deformity (ld) gene disrupt differentiation of the Apical Ectodermal Ridge (AER) and patterning of distal limb structures. However, initial outgrowth of the limb bud is not affected, suggesting that early and late functions of the AER are uncoupled. Similarly, activation of the 5' members of the HoxD gene cluster (Hoxd-11 to Hoxd-13) is not affected in ld mutant posterior limb bud mesenchyme, but the subsequent anteriorization of 5' HoxD domains is delayed by about 12 hours and is associated with reduced levels of polarising activity. These results indicate that the ld gene products act upstream of 5' HoxD genes during patterning of the autopod. Expression of the signalling molecule Sonic hedgehog (Shh) in the posterior limb bud mesenchyme is initiated normally, but ceases prematurely indicating a defect in maintenance of Shh by the ld mutant AER. Furthermore, no Fgf-4 transcripts are detected in the ld mutant AER, whereas Fgf-8 transcripts remain expressed. However, Shh expression can be rescued by heterospecific grafting of ld mutant posterior mesenchyme under a wild-type chicken AER. These studies show that the AER defect in ld homozygous limb buds causes disruption of the FGF-4/SHH feedback loop and support the proposed essential role for FGF-4 in maintaining Shh expression during limb pattern formation.
