Smad4 is required for the normal organization of the cartilage growth plate

Smad4 is the central intracellular mediator of transforming growth factor-beta (TGF-beta) signals. To study the role of Smad4 in skeletal development, we introduced a conditional mutation of the gene in chondrocytes using Cre--loxP system. We showed that Smad4 was expressed strongly in prehypertrophic and hypertrophic chondrocytes. The abrogation of Smad4 in chondrocytes resulted in dwarfism with a severely disorganized growth plate characterized by expanded resting zone of chondrocytes, reduced chondrocyte proliferation, accelerated hypertrophic differentiation, increased apoptosis and ectopic bone collars in perichondrium. Meanwhile, Smad4 mutant mice exhibited decreased expression of molecules in Indian hedgehog/parathyroid hormone-related protein (Ihh/PTHrP) signaling. The cultured mutant metatarsal bones failed to response to TGF-beta1, while the hypertrophic differentiation was largely inhibited by Sonic hedgehog (Shh). This indicated that Ihh/PTHrP inhibited the hypertrophic differentiation of chondrocytes independent of the Smad4-mediated TGF-beta signals. All these data provided the first genetic evidence demonstrating that Smad4-mediated TGF-beta signals inhibit the chondrocyte hypertrophic differentiation, and are required for maintaining the normal organization of chondrocytes in the growth plate.     

IGF signaling directs ventricular cardiomyocyte proliferation during embryonic heart development

Secreted factors from the epicardium are believed to be important in directing heart ventricular cardiomyocyte proliferation and morphogenesis, although the specific factors involved have not been identified or characterized adequately. We found that IGF2 is the most prominent mitogen made by primary mouse embryonic epicardial cells and by a newly derived immortalized mouse embryonic epicardial cell line called MEC1. In vivo, Igf2 is expressed in the embryonic mouse epicardium during midgestation heart development. Using a whole embryo culture assay in the presence of inhibitors, we confirmed that IGF signaling is required to activate the ERK proliferation pathway in the developing heart, and that the epicardium is required for this response. Global disruption of the Igf2 gene, or conditional disruption of the two IGF receptor genes Igf1r and Insr together in the myocardium, each resulted in a significant decrease in ventricular wall proliferation and in ventricular wall hypoplasia. Ventricular cardiomyocyte proliferation in mutant embryos was restored to normal at E14.5, concurrent with the establishment of coronary circulation. Our results define IGF2 as a previously unexplored epicardial mitogen that is required for normal ventricular chamber development.     

A key role for Pak4 in proliferation and differentiation of neural progenitor cells

The Pak4 serine/threonine kinase regulates cytoskeletal organization, and controls cell growth, proliferation, and survival. Deletion of Pak4 in mice results in embryonic lethality prior to embryonic day 11.5. Pak4 knockout embryos exhibit abnormalities in the nervous system, the heart, and other tissues. In this study a conditional deletion of Pak4 was generated in order to study the function of Pak4 in the development of the brain. Nervous system-specific conditional deletion of Pak4 was accomplished by crossing mice with a floxed allele of Pak4 with transgenic mice expressing Cre recombinase under the control of the nestin promoter. The conditional Pak4 knockout mice were born normally, but displayed growth retardation and died prematurely. The brains showed a dramatic decrease in proliferation of cortical and striatal neuronal progenitor cells. In vitro analyses revealed a reduced proliferation and self-renewing capacity of neural progenitor cells isolated from Pak4 knockout brains. The mice also exhibited cortical thinning, impaired neurogenesis and loss of neuroepithelial adherens junctions. By the time the mice died, by 4 weeks after birth, severe hydrocephalus could also be seen. These results suggest that Pak4 plays a critical role in the regulation of neural progenitor cell proliferation and in establishing the foundation for development of the adult brain.     

Essential role of STIM1 in the development of cardiomyocyte hypertrophy

Store-operated Ca²⁺ entry (SOCE) through transient receptor potential (TRP) channels is important in the development of cardiac hypertrophy. Recently, stromal interaction molecule 1 (STIM1) was identified as a key regulator of SOCE. In this study, we examined whether STIM1 is involved in the development of cardiomyocyte hypertrophy. RT-PCR showed that cultured rat cardiomyocytes constitutively expressed STIM1. Endothelin-1 (ET-1) treatment for 48 h enhanced TRPC1 expression, SOCE, and nuclear factor of activated T cells activation without upregulating STIM1. However, the knockdown of STIM1 suppressed these effects, thereby preventing a hypertrophic response. These results suggest that STIM1 plays an essential role in the development of cardiomyocyte hypertrophy.     

Multiple lineage-specific roles of Smad4 during neural crest development

During vertebrate development, neural crest cells are exposed to multiple extracellular cues that drive their differentiation into neural and non-neural cell lineages. Insights into the signals potentially involved in neural crest cell fate decisions in vivo have been gained by cell culture experiments that have allowed the identification of instructive growth factors promoting either proliferation of multipotent neural crest cells or acquisition of specific fates. For instance, members of the TGFβ factor family induce neurogenesis and smooth muscle cell formation at the expense of other fates in culture. In vivo, conditional ablation of various TGFβ signaling components resulted in malformations of non-neural derivatives of the neural crest, but it is unclear whether these phenotypes involved aberrant fate decisions. Moreover, it remains to be shown whether neuronal determination indeed requires TGFβ factor activity in vivo. To address these issues, we conditionally deleted Smad4 in the neural crest, thus inactivating all canonical TGFβ factor signaling. Surprisingly, neural crest cell fates were not affected in these mutants, with the exception of sensory neurogenesis in trigeminal ganglia. Rather, Smad4 regulates survival of smooth muscle and proliferation of autonomic and ENS neuronal progenitor cells. Thus, Smad signaling plays multiple, lineage-specific roles in vivo, many of which are elicited only after neural crest cell fate decision.     

Essential role of the CUL4B ubiquitin ligase in extra-embryonic tissue development during mouse embryogenesis

Mutations of the CUL4B ubiquitin ligase gene are causally linked to syndromic X-linked mental retardation (XLMR). However, the pathogenic role of CUL4B mutations in neuronal and developmental defects is not understood. We have generated mice with targeted disruption of Cul4b, and observed embryonic lethality with pronounced growth inhibition and increased apoptosis in extra-embryonic tissues. Cul4b, but not its paralog Cul4a, is expressed at high levels in extra-embryonic tissues post implantation. Silencing of CUL4B expression in an extra-embryonic cell line resulted in the robust accumulation of the CUL4 substrate p21^Cip1/WAF and G2/M cell cycle arrest, which could be partially rescued by silencing of p21^Cip1/WAF. Epiblast-specific deletion of Cul4b prevented embryonic lethality and gave rise to viable Cul4b null mice. Therefore, while dispensable in the embryo proper, Cul4b performs an essential developmental role in the extra-embryonic tissues. Our study offers a strategy to generate viable Cul4b-deficient mice to model the potential neuronal and behavioral deficiencies of human CUL4B XLMR patients.     

Histone deacetylase 1 controls cardiomyocyte proliferation during embryonic heart development and cardiac regeneration in zebrafish

In contrast to mammals, the zebrafish maintains its cardiomyocyte proliferation capacity throughout adulthood. However, neither the molecular mechanisms that orchestrate the proliferation of cardiomyocytes during developmental heart growth nor in the context of regeneration in the adult are sufficiently defined yet. We identified in a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen the recessive, embryonic-lethal zebrafish mutant baldrian (bal), which shows severely impaired developmental heart growth due to diminished cardiomyocyte proliferation. By positional cloning, we identified a missense mutation in the zebrafish histone deacetylase 1 (hdac1) gene leading to severe protein instability and the loss of Hdac1 function in vivo. Hdac1 inhibition significantly reduces cardiomyocyte proliferation, indicating a role of Hdac1 during developmental heart growth in zebrafish. To evaluate whether developmental and regenerative Hdac1-associated mechanisms of cardiomyocyte proliferation are conserved, we analyzed regenerative cardiomyocyte proliferation after Hdac1 inhibition at the wound border zone in cryoinjured adult zebrafish hearts and we found that Hdac1 is also essential to orchestrate regenerative cardiomyocyte proliferation in the adult vertebrate heart. In summary, our findings suggest an important and conserved role of Histone deacetylase 1 (Hdac1) in developmental and adult regenerative cardiomyocyte proliferation in the vertebrate heart.     

Smad7对Smad2、Smad3、Smad4核转位的抑制作用

研究人永生化支气管上皮BEP2D细胞中,作为Smad蛋白家族的抑制分子,Smad7对TGF-β信号通路中Smad2、Smad3、Smad4核转位的抑制作用.培养BEP2D细胞,瞬时转染Smad7真核表达载体pCISmad7.neo,TGF-β刺激,提取细胞核蛋白及总蛋白,用Western blot方法比较瞬时转染Smad7基因前后细胞核中Smad2、Smad3、Smad4蛋白表达的差异.结果,Smad3在TGF-b作用下有明显的核转位;转染Smad7后Smad3、Smad4的核转位显受到抑制.表明在BEP2D细胞中,Smad7对TGF-β/Smads信号通路的拮抗作用主要通过抑制Smad3的活化、Smad3/Smad4异源复合物的形成及核转位,从而拮抗TGF-β对细胞的生长抑制效应.     

鼻咽癌患者的Smad2/4基因突变分析

利用PCR—SSCP银染技术对30例鼻咽癌患的Smad2／4基因的所有外显子进行突变分析,以探讨Smad2/4基因与鼻咽癌发病的可能相互关系。结果在所有病例的所有外显子上没有发现任何类型的突变。Smad2/4基因可能不是鼻咽癌的易感基因,TGF-β／Smad2/4信号通路可能不参与鼻咽癌的发病。     

Essential mesenchymal role of small GTPase Rac1 in interdigital programmed cell death during limb development

Developing vertebrate limbs are often utilized as a model for studying pattern formation and morphogenetic cell death. Herein, we report that conditional deletion of Rac1, a member of the Rho family of proteins, in mouse limb bud mesenchyme led to skeletal deformities in the autopod and soft tissue syndactyly, with the latter caused by a complete absence of interdigital programmed cell death. Furthermore, the lack of interdigital programmed cell death and associated syndactyly was related to down-regulated gene expression of Bmp2, Bmp7, Msx1, and Msx2, which are known to promote apoptosis in the interdigital mesenchyme. Our findings from Rac1 conditional mutants indicate crucial roles for Rac1 in limb bud morphogenesis, especially interdigital programmed cell death.     

Smad2条件基因剔除载体的构建及LoxP位点有效性鉴定

Smads家族是最新发现的TGF－β信号转导途径中一个重要的新基因家族,SMAD2属于受体激活的SMADs。Smad2在某些肿瘤中发生突变,是一种可能的肿瘤抑制基因。Smad2基因完全剔除小鼠在胚胎期E6．5天死亡,为了研究Smad2在成体各组织器官及肿瘤发生中的可能作用,构建了Smad2条件基因剔除载体,将LoxP置于Smad2基因组序列C末端功能域两侧,并在组成型表达Cre重组酶的大肠杆菌中检测了LoxP位点的功能,该载体的构建为进行Smad2组织特异性基因剔除研究了奠定了基础。     

Establishment of murine Smad5 double knockout ES cells and the studies on their properties

Smad5 is an intracellular transducer of TGF-β signals. Targeted disruption of murineSmad5 gene resulted in embryonic lethal. To study the function ofSmad5 in organgenesis, we generatedSmad5 double knockout ES cells by homologous recombination. We deleted theneo gene of theSmad5 targeted ES cells using Cre-LoxP system.Smad5 double knockout ES cells were obtained by transfecting the targeted ES cells using the same targeting construct. The results of chimeric study showed thatSmad5 might play an important role during the development of heart and neural tube.Smad5 double knockout ES cells formed teratoma when injected subcutaneously into nude mice. They differentiated into several types of cells, including neural cells, muscle cells, chondrocytes, endothelial cells and glandaceous cells.Smad5 double knockout ES cells are useful for studying the function ofSmad5 mediated TGF-β during the organgenesis and thein vitro differentiation of ES cells.     

用酵母双杂交系统研究Smad3和Smad4的相互作用

Ｓｍ ａｄ３ 和 Ｓｍ ａｄ４ 是将 Ｔ Ｇ Ｆ β的信号从细胞外传递到细胞核内的重要的信号传导蛋白． Ｔ Ｇ Ｆ β与其受体结合后,激活受体的磷酸激酶,使 Ｓｍ ａｄ３ 发生磷酸化,活化的 Ｓｍ ａｄ３ 与 Ｓｍ ａｄ４ 结合,形成异源复合物,进入到核中．然后 Ｓｍ ａｄ４ 以 Ｄ Ｎ Ａ 结合蛋白的形式与特定的 Ｄ Ｎ Ａ 结合,将 Ｔ Ｇ Ｆ β的信号传到核内．激活转录,诱导背中胚层的形成,抑制细胞的分化等．经研究利用酵母双杂交试验,鉴定了 Ｓｍ ａｄ３ 和 Ｓｍ ａｄ４ 相互作用的功能区域．构建 Ｓｍ ａｄ３ 和 Ｓｍ ａｄ４ 的 Ｃ 端、 Ｎ 端和中间连接区的突变体,将这些突变体克隆到 ｐ Ｇ Ａ Ｄ４２４ 和 ｐ Ｇ Ｂ Ｔ９ 载体中,并转化到 Ｈ Ｆ７ Ｃ 酵母中．通过 Ｌｅｕ－ ／ Ｔｒｐ－ ／ Ｈｉｓ－ Ｓ Ｄ 平板上菌落的形成,和 Ｘ－ ｇａｌ显色反应鉴定转化到酵母中的两个克隆质粒的相互作用．结果显示 Ｓｍ ａｄ４ 与 Ｓｍ ａｄ３ 异源相五作用时,主要是通过 Ｓｍ ａｄ４ 的中间连接区．在同源作用时, Ｓｍ ａｄ３ 是通过 Ｃ 端,而 Ｓｍ ａｄ４ 是通过中间连接区进行的．     

Establishment of murine Smad5 double knockout ES cells and the studies on their properties

Smad5 is an intracellular transducer of TGF-β signals. Targeted disruption of murine Smad5 gene resulted in embryonic lethal. To study the function of Smad5 in organgenesis, we generated Smad5 double knockout ES cells by homologous recombination. We deleted the neo gene of the Smad5 targeted ES cells using Cre-LoxP system. Smad5 double knockout ES cells were obtained by transfecting the targeted ES cells using the same targeting construct. The results of chimeric study showed that Smad5 might play an important role during the development of heart and neural tube. Smad5 double knockout ES cells formed teratoma when injected subcutaneously into nude mice. They differentiated into several types of cells, including neural cells, muscle cells, chondrocytes, endothelial cells and glandaceous cells. Smad5 double knockout ES cells are useful for studying the function of Smad5 mediated TGF- β during the organgenesis and the in vitro differentiation of ES cells.     

Disruption in the tropomodulin1 (Tmod1) gene compromises cardiomyocyte development in murine embryonic stem cells by arresting myofibril maturation

Tropomodulins (Tmods) comprise a family of capping proteins for actin filament pointed ends. To decipher the significance of Tmod1 functions during de novo myofibrillogenesis, we generated Tmod1 null embryonic stem (ES) cells and studied their differentiation into cardiomyocytes. Strikingly, in vitro cardiomyocyte differentiation of wild type (WT) ES cells faithfully recapitulates in vivo cardiomyocyte differentiation, allowing us to evaluate the phenotypes of Tmod1 knockout (KO) myofibrils irrespective of embryonic lethality of Tmod1 KO mice. Immunofluorescence and electron microscopy studies revealed that Tmod1 null cardiac myocytes were round, morphologically immature, and contained underdeveloped myofibrils that were shorter, narrower, and had fewer thin filaments than those in WT cells. Unexpectedly, clear gaps in the staining pattern for F-actin at the H-zone were detected in most KO cells, indicating the presence of filaments at uniform lengths. This indicates that additional mechanisms other than capping proteins are responsible for thin filament length maintenance in cardiac myocytes. Also unexpectedly, approximately 40% of the KO cardiac myocytes exhibited contractile activity. Our data indicate that differentiating ES cells are a powerful system to investigate the functional properties of contractile proteins and that Tmod1 functions are critical for late stages of myofibrillogenesis, and for the maturation of myofibrils.     

Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor^flox/flox mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function.