ABSTRACT

During the secondary transition of rodent pancreatic development, mainly between E12.5 and E15.5 in mice, exocrine and endocrine populations differentiate from pancreatic progenitors. Here we describe an experimental system for its study in vitro. First, we show that spheres derived from dissociated E12.5 mouse pancreases differentiate within 7 days into most pancreatic exocrine and endocrine cell types, including beta cells. The proportion and spatial repartition of the different endocrine populations mirror those observed during normal development. Thus, dissociation and culture do not impair the developmental events affecting pancreatic progenitors during the secondary transition. Moreover, dissociated cells from mouse E12.5 pancreas were transduced with ecotropic MLV-based retroviral vectors or, though less efficiently, with a mixture of ALV(A)-based retroviral vectors and gesicles containing the TVA (Tumor Virus A) receptor. As an additional improvement, we also created a transgenic mouse line expressing TVA under the control of the 4.5 kB pdx1 promoter (pdx1-TVA). We demonstrate that pancreatic progenitors from dissociated pdx1-TVA pancreas can be specifically transduced by ALV(A)-based retroviral vectors. Using this model, we expressed an activated mutant of the YAP transcriptional co-activator in pancreatic progenitors. These experiments indicate that deregulated YAP activity reduces endocrine and exocrine differentiation in the resulting spheres, confirming and extending previously published data. Thus, our experimental model recapitulates in vitro the crucial developmental decisions arising at the secondary transition and provides a convenient tool to study their genetic control.

摘要

在啮齿动物胰腺发育的二次过渡期间，主要在小鼠的 E12.5 和 E15.5 之间，外分泌和内分泌群体与胰腺祖细胞分化。在这里，我们描述了一个用于体外研究的实验系统. 首先，我们展示了从分离的 E12.5 小鼠胰腺衍生的球体在 7 天内分化为大多数胰腺外分泌和内分泌细胞类型，包括 β 细胞。不同内分泌群体的比例和空间重新分配反映了正常发育过程中观察到的情况。因此，解离和培养不会损害在二次过渡期间影响胰腺祖细胞的发育事件。此外，从小鼠 E12.5 胰腺中分离的细胞用亲嗜性的基于 MLV 的逆转录病毒载体进行转导，或者用基于 ALV(A) 的逆转录病毒载体和含有 TVA（肿瘤病毒 A）受体的凝胶的混合物转导，尽管效率较低。作为额外的改进，我们还创建了一个在 4.5 kB pdx1控制下表达 TVA 的转基因小鼠系。启动子（pdx1 -TVA）。我们证明来自分离的pdx1 -TVA 胰腺的胰腺祖细胞可以通过基于 ALV(A) 的逆转录病毒载体进行特异性转导。使用该模型，我们在胰腺祖细胞中表达了 YAP 转录共激活因子的激活突变体。这些实验表明，失调的 YAP 活动减少了所得球体中的内分泌和外分泌分化，证实并扩展了先前公布的数据。因此，我们的实验模型在体外概括了在二次转变中出现的关键发育决定，并为研究它们的遗传控制提供了一个方便的工具。