RESUMO
Intestinal stem cells (ISCs) are maintained by a niche mechanism, in which multiple ISCs undergo differential fates where a single ISC clone ultimately occupies the niche. Importantly, mutations continually accumulate within ISCs creating a potential competitive niche environment. Here we use single cell lineage tracing following stochastic transforming growth factor ß receptor 2 (TgfßR2) mutation to show cell autonomous effects of TgfßR2 loss on ISC clonal dynamics and differentiation. Specifically, TgfßR2 mutation in ISCs increased clone survival while lengthening times to monoclonality, suggesting that Tgfß signaling controls both ISC clone extinction and expansion, independent of proliferation. In addition, TgfßR2 loss in vivo reduced crypt fission, irradiation-induced crypt regeneration, and differentiation toward Paneth cells. Finally, altered Tgfß signaling in cultured mouse and human enteroids supports further the in vivo data and reveals a critical role for Tgfß signaling in generating precursor secretory cells. Overall, our data reveal a key role for Tgfß signaling in regulating ISCs clonal dynamics and differentiation, with implications for cancer, tissue regeneration, and inflammation.
Assuntos
Diferenciação Celular/genética , Celulas de Paneth/citologia , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Células-Tronco/citologia , Animais , Linhagem da Célula/genética , Rastreamento de Células , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citologia , Camundongos , Mutação , Celulas de Paneth/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Transdução de Sinais , Análise de Célula Única/métodos , Nicho de Células-Tronco/genética , Células-Tronco/metabolismoRESUMO
The DNA mismatch repair (MMR) machinery in mammals plays critical roles in both mutation avoidance and spermatogenesis. Meiotic analysis of knockout mice of two different MMR genes, Mlh1 and Mlh3, revealed both male and female infertility associated with a defect in meiotic crossing over. In contrast, another MMR gene knockout, Pms2 (Pms2(ko/ko)), which contained a deletion of a portion of the ATPase domain, produced animals that were male sterile but female fertile. However, the meiotic phenotype of Pms2(ko/ko) males was less clear-cut than for Mlh1- or Mlh3-deficient meiosis. More recently, we generated a different Pms2 mutant allele (Pms2(cre)), which results in deletion of the same portion of the ATPase domain. Surprisingly, Pms2(cre/cre) male mice were completely fertile, suggesting that the ATPase domain of Pms2 is not required for male fertility. To explore the difference in male fertility, we examined the Pms2 RNA and found that alternative splicing of the Pms2(cre) allele results in a predicted Pms2 containing the C-terminus, which contains the Mlh1-interaction domain, a possible candidate for stabilizing Mlh1 levels. To study further the basis of male fertility, we examined Mlh1 levels in testes and found that whereas Pms2 loss in Pms2(ko/ko) mice results in severely reduced levels of Mlh1 expression in the testes, Mlh1 levels in Pms2(cre/cre) testes were reduced to a lesser extent. Thus, we propose that a primary function of Pms2 during spermatogenesis is to stabilize Mlh1 levels prior to its critical crossing over function with Mlh3.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fertilidade , Proteínas Nucleares/metabolismo , Deleção de Sequência , Testículo/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina Trifosfatases/genética , Motivos de Aminoácidos , Animais , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 1 Homóloga a MutL , Proteínas Nucleares/genética , Testículo/fisiologiaRESUMO
We developed a cell division-activated Cre-lox system for stochastic recombination of loxP-flanked loci in mice. Cre activation by frameshift reversion is modulated by DNA mismatch-repair status and occurs in individual cells surrounded by normal tissue, mimicking spontaneous cancer-causing mutations. This system should be particularly useful for delineating pathways of neoplasia, and determining the developmental and aging consequences of specific gene alterations.
Assuntos
Adenosina Trifosfatases/genética , Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Integrases/genética , Envelhecimento/genética , Animais , Mutação da Fase de Leitura , Genes ras/genética , Intestinos/enzimologia , Camundongos , Endonuclease PMS2 de Reparo de Erro de Pareamento , Neoplasias/etiologia , Neoplasias/genética , Recombinação Genética , beta-Galactosidase/genéticaRESUMO
Early in development, neurons only express NR1/NR2B-containing N-methyl-d-aspartate (NMDA) receptors. Later, NR2A subunits are upregulated during a period of rapid synapse formation. This pattern is often interpreted to indicate that NR2A-containing receptors are synaptic and that NR2B-containing receptors are extrasynaptic. We re-examined this issue using whole cell recordings in cultured hippocampal neurons. As expected, the inhibition of whole cell currents by the NR2B-specific antagonist, ifenprodil, progressively decreased from 69.5 +/- 2.4% [6 days in vitro (DIV)] to 54.9 +/- 2.6% (8 DIV), before reaching a plateau in the second week (42.5 +/- 2%, 12-19 DIV). In NR2A-/- neurons, which express only NR1/NR2B-containing NMDA receptors, autaptic excitatory postsynaptic currents (EPSCs; > or =12 DIV) were more sensitive to ifenprodil and decayed more slowly than EPSCs in wild-type neurons. Thus NR2B-containing receptors were not excluded from synapses. We blocked synaptic NMDA receptors with MK-801 during evoked transmitter release, thus allowing us to isolate extrasynaptic receptors. Ifenprodil inhibition of this extrasynaptic population was highly variable in different neurons. Furthermore, extrasynaptic receptors in autaptic cultures were only partially blocked by ifenprodil, indicating that NR2A-containing receptors are not exclusively confined to the synapse. Extrasynaptic NR2A-containing receptors were also detected in NR2A(-/-) neurons transfected with full-length NR2A. Truncation of the NR2A C terminus did not eliminate synaptic expression of NR2A-containing receptors. Our results indicate that NR2A- and NR2B-containing receptors can be located in either synaptic or extrasynaptic compartments.
