RESUMEN
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.
Asunto(s)
Diferenciación Celular/genética , Células de Paneth/citología , Proteínas Serina-Treonina Quinasas/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Células Madre/citología , Animales , Linaje de la Célula/genética , Rastreo Celular , Regulación del Desarrollo de la Expresión Génica , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citología , Ratones , Mutación , Células de Paneth/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Transducción de Señal , Análisis de la Célula Individual/métodos , Nicho de Células Madre/genética , Células Madre/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/genética , Reparación de la Incompatibilidad de ADN , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Integrasas/genética , Envejecimiento/genética , Animales , Mutación del Sistema de Lectura , Genes ras/genética , Intestinos/enzimología , Ratones , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Neoplasias/etiología , Neoplasias/genética , Recombinación Genética , beta-Galactosidasa/genéticaRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , Reparación de la Incompatibilidad de ADN , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Fertilidad , Proteínas Nucleares/metabolismo , Eliminación de Secuencia , Testículo/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Adenosina Trifosfatasas/genética , Secuencias de Aminoácidos , Animales , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Expresión Génica , Masculino , Ratones , Ratones Noqueados , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Homólogo 1 de la Proteína MutL , Proteínas Nucleares/genética , Testículo/fisiologíaRESUMEN
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.