The Nuclear Envelope in Development, Disease and Ageing

脊椎动物核层是一种与内膜核面相关的蛋白质网。它为染色质结构域提供锚定位点，是间期核结构、DNA复制和染色质组织的重要决定因素。层的主要成分是中间的丝状蛋白，即核层蛋白，层蛋白分为a型和b型两类。b型层蛋白由2个基因编码并组成表达，而a型层蛋白是由单个基因（Lmna）拼接而成的变体，其表达受发育调控。它们在早期胚胎或成体干细胞中不表达，其表达与各种谱系的终末分化有关。我们得到了不表达a型层粘连蛋白的小鼠。Lamin缺失小鼠的发育表面上是正常的，但在4-5周时，它们出现了严重的肌肉萎缩症，心脏异常，并在8周时死亡。在人类中，不同的Lamin A基因突变导致了至少8种遗传性疾病。包括两种形式的肌肉萎缩症，扩张型心肌病，两种类型的家族性部分脂肪营养不良症，其中一种还影响骨骼发育，周围神经病变，以及最近的一种早衰症，叫做哈钦森-吉尔福德早衰症。我们已经获得了与大多数这些疾病相对应的Lmna基因突变的小鼠。我们已经生产出肌肉萎缩、扩张型心肌病和早衰表型的小鼠系。患有早衰症的小鼠为衰老的基本方面以及细胞核结构如何调节细胞增殖提供了新的见解。此外，我们设计了一种突变小鼠系，其中含有另一种Lamin a /C蛋白的异构体，称为N195K点突变，与人类扩张型心肌病（DCM）病例相关。虽然纯合子N195K/N195K小鼠似乎比最初获得的Lamin A/C缺失的动物寿命更长，没有出现明显的肌肉萎缩和脂肪损失，但这些动物最终在4个月大时死于急性心力衰竭，并伴有心律失常，类似于人类DCM患者的临床发现。我们还能够通过传统的loxP/Cre系统获得一种允许有条件地消除Lamin a /C基因的小鼠系。这些动物提供了一种替代方法，可以通过与表达cre的转基因细胞系杂交，以诱导的方式研究特定组织亚群中的核层功能，从而绕过在Lamin a /C缺失小鼠中观察到的早期致命性。我们利用这一小鼠品系在实验室中研究了Lamin A/C缺失对毛发周期的影响（通过与K14-Cre转基因系杂交），以及对造血干细胞所在的骨生态位环境中免疫系统细胞成分成熟的影响（通过与Col2a-Cre转基因小鼠交配）。总之，对椎板病的分析为细胞核结构对其功能的重要性提供了新的信息。这是特别相关的，因为除了与层蛋白相关的疾病（层蛋白病）外，至少还有两种其他疾病与与核膜相关的蛋白质突变有关。此外，最近对核膜成分的蛋白质组学分析表明，另外14种疾病也可能与核膜蛋白的改变有关。除了这些疾病外，我们还在研究层粘连蛋白缺乏对细胞复制DNA、染色体分离/定位和染色质组织/基因调控的影响，以及层粘连蛋白缺失是否会影响肿瘤的发展。

The vertebrate nuclear lamina is a protein meshwork associated with the nuclear face of the inner nuclear membrane (INM). It provides anchoring sites for chromatin domains, and is an important determinant of interphase nuclear architecture, DNA replication and chromatin organization. The major components of the lamina are the intermediate filament-like proteins, the nuclear lamins, The lamins are grouped into 2 classes, A-type and B-type. The B-type lamins are encoded by 2 genes and are constitutively expressed whereas the A-type lamins are spliced variants from a single gene (Lmna) and their expression is developmentally regulated. They are not expressed in early embryos or adult stem cell and their expression correlates with the terminal differentiation of various lineages. We derived mice that did not express the A-type lamins. Development of the Lamin null mice was overtly normal, but by 4-5 weeks they developed a severe form of muscular dystrophy, had abnormal hearts and were dead by 8 weeks. In humans different mutations in the Lamin A gene are responsible for at least 8 inherited diseases. These include 2 forms of muscular dystrophy, dilated cardiomyopathy, 2 types of Familial Partial Lipodystrohy, one of which also affects skeletal development, a peripheral neuropathy and most recently, the premature ageing condition called Hutchinson Gilford Progeria. We have derived mice with mutations in the Lmna gene that correspond to most of these diseases. We have produced mouse lines that develop muscular dystrophy, dilated cardiomyopathy, and a progeric phenotype. The mice with progeria are providing novel insights into fundamental aspects of aging and how the structure of the nucleus developmentally regulates cell proliferation. In addition, we have engineered a mutant mouse line harboring yet another isoform of Lamin A/C protein, known as N195K point mutation and associated in humans with cases of dilated cardiomyopathy (DCM). Although the homozygous N195K/N195K mice appeared to live longer and developed no visible muscular wasting of fat loss compared to the initially derived Lamin A/C null animals, these animals ultimately succumb by the age of 4 months due to acute heart failure preceded by cardiac aberrant rhythm similar to the clinical findings in human DCM patients. We were also able to derive a mouse line allowing for a conditional elimination of Lamin A/C gene via a conventional loxP/Cre system. These animals provide an alternative of studying the nuclear lamina functioning in specific subsets of tissues and/or in an inducible manner by crossing with a spectrum of Cre-expressing transgenic lines thus bypassing the early lethality observed in Lamin A/C null mice. We exploited this mouse strain in the lab to study the effect of Lamin A/C loss on the hair cycle (via crossing with K14-Cre transgenic line) and on the maturation of immune system cellular components in the bone niche environment harboring the hematopoietic stem cells (via mating with Col2a-Cre transgenic mice). In summary, the analysis of the laminopathies is providing novel information into how the structure of the nucleus is important to its function. This is particularly relevant, as in addition to the diseases associated with the lamins (the laminopathies), at least 2 other disease have been linked to mutations in proteins associated with the nuclear envelope. Furthermore, a recent proteomics analysis of the components of the nuclear envelope suggested that an additional 14 diseases might also be linked to altered nuclear envelope proteins. In addition to these diseases we are also looking at what effects Lamin deficiency has on a cells ability to replicate its DNA, chromosome segregation/location and chromatin organization/gene regulation and whether lamin loss may affect tumor development.