RESUMO
FGF signaling is essential for mammary gland development, yet the mechanisms by which different members of the FGF family control stem cell function and epithelial morphogenesis in this tissue are not well understood. Here, we have examined the requirement of Fgfr2 in mouse mammary gland morphogenesis using a postnatal organ regeneration model. We found that tissue regeneration from basal stem cells is a multistep event, including luminal differentiation and subsequent epithelial branching morphogenesis. Basal cells lacking Fgfr2 did not generate an epithelial network owing to a failure in luminal differentiation. Moreover, Fgfr2 null epithelium was unable to undergo ductal branch initiation and elongation due to a deficiency in directional migration. We identified FGF10 and FGF2 as stromal ligands that control distinct aspects of mammary ductal branching. FGF10 regulates branch initiation, which depends on directional epithelial migration. By contrast, FGF2 controls ductal elongation, requiring cell proliferation and epithelial expansion. Together, our data highlight a pleiotropic role of Fgfr2 in stem cell differentiation and branch initiation, and reveal that different FGF ligands regulate distinct aspects of epithelial behavior.
Assuntos
Epitélio/crescimento & desenvolvimento , Fator 10 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Morfogênese , Animais , Animais Recém-Nascidos , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ligantes , Glândulas Mamárias Animais/crescimento & desenvolvimento , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Fosfatidilinositol 3-Quinases/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Regeneração/efeitos dos fármacos , Regeneração/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.
Assuntos
Alelos , Proteína Huntingtina/genética , Doença de Huntington/terapia , Mutação , Transcrição Gênica , Dedos de Zinco , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Doença de Huntington/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Neuroproteção , Repetições de TrinucleotídeosRESUMO
Fibroblast growth factor (FGF) signaling is essential for vertebrate organogenesis, including mammary gland development. The mechanism whereby FGF signaling is regulated in the mammary gland, however, has remained unknown. Using a combination of mouse genetics and 3D ex vivo models, we tested the hypothesis that Spry2 gene, which encodes an inhibitor of signaling via receptor tyrosine kinases (RTKs) in certain contexts, regulates FGF signaling during mammary branching. We found that Spry2 is expressed at various stages of the developing mammary gland. Targeted removal of Spry2 function from mammary epithelium leads to accelerated epithelial invasion. Spry2 is up-regulated by FGF signaling activities and its loss sensitizes mammary epithelium to FGF stimulation, as indicated by increased expression of FGF target genes and epithelia invasion. By contrast, Spry2 gain-of-function in the mammary epithelium results in reduced FGF signaling, epithelial invasion, and stunted branching. Furthermore, reduction of Spry2 expression is correlated with tumor progression in the MMTV-PyMT mouse model. Together, the data show that FGF signaling modulation by Spry2 is essential for epithelial morphogenesis in the mammary gland and it functions to protect the epithelium against tumorigenesis.
Assuntos
Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Morfogênese , Transdução de Sinais , Animais , Cruzamentos Genéticos , Modelos Animais de Doenças , Feminino , Heterozigoto , Humanos , Integrases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Vírus do Tumor Mamário do Camundongo/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Serina-Treonina QuinasesRESUMO
Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.