national resource center for mutant mice
model animal research center of nanjing university
moe key laboratory of model animal for disease study nanjing biomedical research institute of nanjing university
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director’s Words
S everal years ago, I went out to climb Xiangshan (The Fragrant Mountain) at Beijing with a good friend of mine who just published an interesting work in Cell. It was a big deal then.
He told me that publishing is like hiking. In the bottom of the mountain, you are all geared up and have full confidence that you will reach the summit. However, you may find out it is really not easy at the half way. In addition to the sweating body and sore muscles, you may also start to doubt the meaning of getting to the top. After all, this mountain might only present the similar ecological landscape and you are able to enjoy this beauty already. “So it is like you published some nice data on JBC every year and you felt quite comfortable”, my friend said.
However, “to getting to the top is not only for seeing more of this mountain or conquering the self-pity, but to have a different viewpoint for the world”.How many of us really enjoy the research processes and understand our destinies as scientists?
MARC is 8 years old now and we are in the half way to the summit. This year the blooming of publications from many laboratories rewarded the hard work of our research teams. I was very happy to know Dr. Yang’s paper on PDK1-Akt signaling role in heart development to be selected as spotlight article of MCB, as well as Dr.He’s paper on dissecting VEGFR3 function on lymphangiogenesis and angiogenesis was selected as cover of recent CR. My congratulations also extend to Dr.Xu and Dr.Chen, who get their first paper published as correspondence author.
However, we are still having a long way to reach the summit. The key issue is that we still need to look for the true novelty and ingenuity in our research. Luckily, 2010 gave us more confidence to work even harder. And we will get there.
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Director’s words
Management Structure of MARC Research Laboratories Ying Cao Jiong Chen Xiang Gao Jun Gao Yulong He Xingxu Huang Qing Jiang Chaojun Li Geng Liu Jianghuai Liu Cheng Sun Ying Xu Jun Yan Zhongzhou Qingshun Zhao Mingsheng Zhu Qing Zhang Student of the year 2010
National Resource Center for Mutant Mice Lab Members 2010 Summer School 2010 Students Union 2010 Seminar
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34-56-78-910-1112-1314-1516-1718-1920-2122-2324-2526-2728-2930-3132-33343536-3940-4344-454647management structure of model animal research center (marc )
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Xenopus embryogenesis
Ying Cao Ph.D.
Cao Ying made the PhD study at the University of Essen, Germany, from 1998 to 2002. During the period he performed a screening of novel genes involved in the early embryogenesis of Xenopus laevis and identified a few new genes that play essential roles in Xenopus embryonic development. In 2002, he earned PhD degree and graduated summa cum laude because of the extraordinary performance during PhD study. Afterwards during the years from 2002 to 2008 he joined the Institute of Biochemistry, University of Ulm, Germany, and continued the study on Xenopus development, especially on the molecular mechanisms underlying germ layer formation. From October 2008, he was offered the professor at MARC and set up the laboratory for Xenopus developmental biology.
G
erm layer formation is one of the most important events during early embryogenesis. It includes many coordinated regulated signals, which determines the cell fates and movements such that the germ layers can be formed in the correct temporal and spatial patterns. We in the former time have investigated the function of Oct25, one of the Oct4 homologous proteins in Xenopus, during Xenopus development, and found that it inhibits mesoderm and endoderm germ layer formation via blocking the functions of the signals that promote germ layer formation, notably maternal VegT, β-catenin and zygotic nodal. Recently we found that the POU domain in the Oct25 protein is indispensible for its function, for disruption of the POU domain structure caused functional reversal of the protein. This was supported by the fact that overexpression of the Oct25 mutant in Xenopus embryos caused partial secondary axis formation while the wild type protein inhibited axis formation (Figure 1). The effect might also suggest an important molecular mechanism underlying the function of Oct25 during germ layer formation. We are currently trying to analyze why the POU domain is crucial for Oct25. Albeit quite many studies have shown that Oct4 is transcriptionally regulated by other pluripotency factors, it is not known whether it is regulated by differentiation signals. We found that the three Xenopus Oct4 homologous genes Oct60, Oct25 and Oct91 are differentially regulated by FAST1 (Figure 2), a transcription factor in the nodal pathway. Detailed mechanism for this effect is under investigation.
Contact Information
Tel.: +86-25-58641537 (Office) +86-25-58641539 (Lab) Fax: +86-25-58641500
Email: caoying@https://www.doczj.com/doc/e51992939.html, yyy_cao@https://www.doczj.com/doc/e51992939.html,
Fig. 1
The smaller image (upper left) denotes the wild type neurula embryo with normal neural fold, the bigger (lower right) denotes the neurula embryo expressing the mutant Oct25 develops bifurcated neural folds, one is primary and the other is induced secondary neural fold.
Fig. 2
FAST1 overexpression in Xenopus embryos regulates transcription of Oct4 homologous genes.
Lab members
Graduate Students
? Lei Lu Qing Cao Wei Zhao Yan Gao
Technician
? Xuena Zhang Haihua Ma
Control
Oct60
Oct25
Oct91
FAST1 injection
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actin dynamics in development
Jiong Chen Ph.D.
Jiong Chen received his Bachelor in Biochemistry (1995) and his Ph.D. in Molecular, Cell and Developmental Biology (2001), both from University of California, Los Angeles (UCLA). His Ph.D. thesis was carried out in Frank Laski’s lab and it was focused on the genetics and developmental studies of cell movement processes in the Drosophila ovary. From 2002 to 2004, Jiong did his postdoctoral research in Drosophila eye development under the guidance of Utpal Banerjee at UCLA, and it was combined with an undergraduate teaching experience that was funded by a HHMI teaching/research grant. He joined the Faculty of Model Animal Research Center (MARC), Nanjing University in 2004. He is now a professor of genetics and developmental biology and a principle investigator in MARC.
M
y lab is mainly interested in how actin dynamics regulate such essential cellular and developmental processes as cell migration and epithelial polarity and morphogenesis. Among the major players that modulate the dynamics of actin cytoskeleton is the actin depolymerizing factor-cofilin, which promotes rapid turnover and reassembly of actin filaments. My lab is currently studying the in vivo roles of cofilin, its cofactor-actin interacting protein 1 (AIP1), and another G-actin binding protein-twinfilin in a developmental context. The functions of these essential actin dynamics regulators had been mostly determined in test tubes or in cell cultures, but how they actually function physiologically and in a complex system such as in epithelial morphogenesis or chemotaxis of a group of cells within tissues are less understood. My lab has employed a mainly genetic and functional genomics approach, using the model animal Drosophila melanogaster and cell biological techniques to conduct most of the experiments. And there are two model systems that we mainly use in the lab: border cell migration in the Drosophila ovary and the eye epithelium of Drsophila larvae and pupae (Fig. 1, 2). Border cell migration is an excellent in vivo and genetically tractable system to study molecular mechanism underlying guided migration or chemotaxis, and the tumor-like invasive migration of border cells through large germline tissues can also be used as a model to identify novel genes essential for cancer metastasis as well as cell migration in development (Fig. 1). In addition, since 6-10 border cells always migrate as a coherent cluster, it has been recently used as a model system to study collective cell migration, which is prevalent in morphogenesis, cancer and regeneration. Currently, we are interested in the following three questions. 1. How extracellular factors (gradients) guide the cluster of border cells? What novel guidance receptors and signaling (besides PVR and EGFR) are involved? 2. How signaling pathways affect actin cytoskeleton? And through what actin dynamics regulators? 3. Are there novel regulatory mechanisms that link other important cellular process with cell migration? Below is a list of three projects (1-3) ongoing in the lab to address these questions.
The single layer of developing eye epithelium (eye imaginal disc) is a
Lab members
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Tel.: +86-25-58641507 (Office) +86-25-58641509 (Lab) Fax: +86-25-58641500 Email: chenjiong@https://www.doczj.com/doc/e51992939.html,
system that we used to probe the roles of actin dynamics regulators in epithelial morphogenesis (Fig 3). We are currently studying AIP1’s roles in adherens junction (AJ) rearrangement and maintenance during epithelial morphogenesis of ommatidial pre-clusters (Project 4 below). We are also using tissue cell cultures and AIP1 conditional knockout mouse to address if AIP1’s roles in epithelial morphogenesis are conserved in mammals (Project 6, in collaboration with Dr. Zhongzhou Yang’s lab). In addition, we are investigating each of Rho GTPases’ (Rho, Rac and Rac) distinct roles in epithelial morphogenesis: proliferation, neuronal cells specification, and terminal differentiation (Project 5).
Fig. 1 Cofilin is required for collective migration of border cells in Drosophila
Graduate Students
? Dandan Chu Di Kang Jun Luo Hanshuang Pan Ping Wan Jing Wu
Technical Staff ?
Hong Zhu
Fig. 2 AIP1 promotes actin dynamics and regulates the remodeling of adherens junctions in the developing eye epithelium of Drosophila
Below is a list of projects currently going on in the lab.
1. Regulation of cofilin phosphorylation asymmetry during chemotaxis of border cells in Drosophila ovary. (Fig. 1)
2. The roles of novel intracellular traffic regulators Dlg5 and Blot in border cell migration and junctional regulation in follicle epithelial cells.
3. Clonal and RNAi screens in border cells to identify novel genes and signaling pathways that are essential for cell migration and chemotaxis.
4. AIP1’s role in adherence junction (AJ) rearrangement and maintenance during epithelial morphogenesis in Drosophila eye development. (Fig. 2)
5. Rho, Rac, and Cdc42 each plays distinct functions in different stages of eye epithelial morphogenesis.
6. Establish mouse models with AIP1 conditional knockout.
7. Cofilin and AIP1’s roles in bristle morphogenesis.
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6
Xiang Gao Ph.D.
Xiang received his Ph.D. degree of Anatomy and Development Biology at Thomas Jefferson University in 1994. After postdoctoral training at Roche Institute of Molecular Biology and The Jackson laboratory, as alumni of Nanjing University, he was recruited back as professor of genetics at Institute of Molecular Medicine at year 2000. Two years later Xiang become the founder for both National Resource Center for Mutant Mice (NRCMM) and Model Animal Research Center (MARC). Currently he is the director for MARC and Chairperson for NRCMM.
genetic manipulation and disease models
T
he broad biological questions our laboratory wishes to answer are the molecular mechanisms for controlling physiological homeostasis, as well as the causes for deleterious diseases related to such mechanisms. Genetic manipulation is the key technology we use for defining the novel pathways and regulatory genes. For instance, our laboratory is interest in understanding the in vivo function of phosphatase PP2A. As one of the most abundant protein phosphatases in animal cells, PP2A has been characterized extensively in vitro. However, the role of PP2A in normal physiological homeostasis remains unknown due to lack of animal models. We have generated conditional targeted mice for two genes encoding PP2A catalytic subunits (Cα and C?). Phenotyping data not only suggested Cα and C? are functional distinguished in different tissues (though both of them are ubiquitously expressed in all types of cells and indistinguishable in vitro assay), but suggested PP2A involves distinct signaling pathways in different cell types. For example, mice lacking PP2ACα in myocardial cells displayed heart hypertrophy. However, knockout the same gene in hepatocyte induced liver cancer. Moreover, while our data indicated C? was dispensable for most physiological processes due to compensatory functions of Cα, but it is required for germ cell differentiation. Hopefully, by systematically analyzing the alteration of signaling networks in various tissues lacking PP2A activity, we will define novel regulatory interaction and Lab members
Research Associate
? Shiying Guo Xiaoqing Yuan Yao Li Jing Tang
Graduate students
? Weiqian Chen Jingyue Xu Xin Qi Pengyu Gu Yue Zhou Xuan Jiang Zan Huang Xin Tu Li Xian Shiyuan Hou An Tang Anying Song
LCNS I.D. length % identity Chr. 5' gene distance
3' gene distance
11 546 95.6 chr1 Mfsd9 637kb Pou3f3 1269kb 12 543 95.8 chr1 Mfsd9 Pou3f3 13 513 96.3 chr1 Mfsd9 Pou3f3 54 639 96.2 chr2 Zeb2 2221kb Acvr2a 1479kb 64 677 96.5 chr2
Zeb2
Acvr2a
(up to 39)
≥ 500
≥ 95
>500kb >500kb Contact Information
Tel.: +86-25-58641598 (Office) +86-25-58641511 (Lab) Fax: +86-25-58641500 Email: gaoxiang@https://www.doczj.com/doc/e51992939.html,
function of different signaling pathways as well the novel mechanism for various diseases.
In addition, we are also interested in the chromatin architecture modulation that affects coordinated gene regulation in the whole genome level and sophisticated epigenetic control. we are keen to understanding the function of some conserved non-coding sequence in mammalian genome. By comparative analysis of genomes from mouse to human, more than 400 segments with following categories, length longer than 500bp and homology higher than 95%, were identified. We name them as LCNS (long conserved non-coding sequence). Furthermore, bioinformatic study indicated more than 10% of them are located in “gene desert” area, defined by absence of coding genes within 500kb of LCNS. Our earlier work indicated that these regions could be crucial to regulate the gene expression. For instance, about 870kb upstream of BDNF gene contains several genome fragments which are important for the neuronal specific expression of BDNF. Disruption of these fragments led to dramatic decrease of expression and caused the type II diabetes both in mice and human. Therefore, we are trying to define the physiological and molecular role of these LCNS by generating knockout mice systematically.
Example of some of the Long Conserved Non-coding Sequences (LCNS).
A B C
Fig. 1 Obesity, hyperphagia and increased linear growth in Timo mice.
A: severely obese Timo/Timo mouse and moderately obese Timo/+ mouse at at 24 wk of age.
B: measurement of daily food intake of 12-wk-old female mice.
C: increased linear growth in both Timo/Timo and Timo/+ mice.
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ageing and disease
Jun Gao Ph.D.
Jun Gao received her Ph.D. from the University of Science and Technology of China (2004). Her Ph.D. thesis was carried out in Dr Tian-Le Xu’s lab and it was focused on the function of ion channels and their roles in disease. After postdoctoral training at the Institute of Neuroscience of Shanghai and the Picower institute for learning and memory in Massachusetts Institute of Technology, she joined the Faculty of Model Animal Research Center (MARC), Nanjing University. She is now an associated professor and a principle investigator in MARC.
T
he NAD-dependent deacetylase Sir2 was initially identified as a mediator of replicative lifespan in budding yeast and was subsequently shown to modulate longevity in worms and flies. Its mammalian homologue, SIRT1, appears to have evolved complex systemic roles in cardiac function, DNA repair, and genomic stability. Recent studies suggest a functional relevance of SIRT1 in normal brain physiology and neurological disorders. However, it is unknown if SIRT1 plays a role in higher-order brain functions. Here we show that activation of SIRT1, the Sir2 homolog in mice, by resveratrol resulted in memory facilitation in mice (Fig. 2A). To directly evaluate the physiological role of Sirt1 in the brain, Sirt1 mutant mice (Sirt1Δ, lacks part of the catalytic domain) were generated by crossing mice carrying a floxed Sirt1Δex4 allele with Nestin-Cre transgenic mice. Remarkably, the Sirt1Δ mice showed markedly decreased freezing behaviour as evaluated by the context- and tone-dependent fear conditioning paradigm 24hr after training when compared to Cont littermates (Fig. 2A). Consistent with it, the enhancement effect of Res on learning was not observed in Sirt1Δ mice (Fig. 2A). Thus, Sirt1 loss of function impaired associative learning. To further evaluate the integrity of hippocampus-dependent memory formation in the Sirt1 mice, we utilized the Morris water maze paradigm. Sirt1Δ mice showed significantly enhanced escape latency throughout the
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Tel.: +86-25-58641546(Office) Fax: +86-25-58641500 Email: gaojun@https://www.doczj.com/doc/e51992939.html,
training process (Fig.2B). Since the synapse is well known to be the cellular basis for learning and memory, we investigated the role of Sirt1 on the density of dendritic spines synapse numbers. As shown in Fig. 2C and D, neuron-specific expression of a mutant SIRT1 protein that lacks part of the catalytic domain, reduced dendritic spine density and synapse number. Moreover, in SIRT1 mutant mice, the level of BDNF and CREB in hippocampus was significantly lower than that in control mice. Surprisingly, these effects were mediated via post-transcriptional regulation of CREB expression by a brain-specific microRNA, miR-134. SIRT1 normally functions to limit expression of miR-134 via a repressor complex containing the transcription factor YY1, and unchecked miR-134 expression following SIRT1 deficiency results in the down-regulated expression of CREB and BDNF, thereby impairing synaptic plasticity (Fig. 1). These findings demonstrate a novel role for SIRT1 in cognition and a previously unknown microRNA-based mechanism by which SIRT1 regulates these processes. Furthermore, these results describe a separate branch of SIRT1 signaling, in which SIRT1 has a direct role in regulating normal brain function in a manner that is disparate from its cell survival functions, demonstrating its value as a potential therapeutic target for the treatment of CNS disorders.
Technical Staff ? Yuehua Xiao
Graduate Student ? Beibei Lai
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Fig.3 SIRT1Δ mice display impaired LTP .
Fig.2 SIRT1 loss-of-function impairs memory and synaptic plasticity
(A) Freezing behavior 24 hr after training is reduced in SIRT1Δ mice compared with their floxed littermates controls (Cont) (SIRT1Δ, n = 13; Cont, n = 15, **p<0.01 ). and SIRT1Δ animals given Resveratrol (Res) treatment ICV for a week do not display enhanced freezing behavior in contrast to control mice (SIRT1Δ, n = 12; Cont, n = 16, *p<0.05; **p<0.01 ).
(B) The Morris water maze hidden platform test. Escape latencies of control mice improved significantly faster than that of SIRT1Δ mice (SIRT1Δ, n = 9; Cont, n=10; **p<0.01). Right panel: representative path tracings of the probe test on day 5. (C) Synaptophysin (SVP) immunoreactivity is reduced in SIRT1Δ hippocampi (SIRT1Δ, n = 23 slices, 3 mice; Cont, n = 23 slices, 3 mice, ***p<0.001).
(D) Hippocampal neuronal dendritic spine density is reduced in SIRT1Δ mice (SIRT1Δ, n = 22; Cont, n = 26, **p<0.01).
(A) LTP was induced by two theta-burst stimulations (TBS) in the CA1 region of acute slices from 5~6 month old SIRT1Δ mice or their floxed littermates controls. Forty minutes post-stimulation, fEPSPs from SIRT1Δ mice had decayed to the baseline (n = 8 slices, 108.5± 3.2% compared with baseline), whereas fEPSPs from control mice remained potentiated (n= 9 slices, 162.0 ± 14.7% compared with baseline).
(B and C) Resveratrol facilitates LTP in a SIRT1-dependent manner. LTP was induced by one TBS in the CA1 region from control or SIRT1Δ acute slices with or without Res (5 μM) pretreatment for 1 hour. Slices from control mice showed increased LTP formation under Res treatment (B; n = 6 slices, 132.2 ± 7.8% compared with baseline), at 50 min after
induction, whereas slices from SIRT1Δ mice did not show enhanced LTP with Res treatment (C; n = 8 slices, 109.2 ± 10.7% compared with baseline)
A A C C
D
B
B
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vascular and cancer biology
Yulong He Ph.D.
Yulong He received his Ph.D. in Molecular Biology from University of Cambridge in 1999. He did his first Postdoctoral training in the Department of Pathology, University of Cambridge, from Apr. 1999 to Nov. 2000. Then he moved to Molecular Cancer Biology Laboratory, University of Helsinki, and worked as a Postdoctoral researcher from Nov. 2000 to Jul. 2003. He was employed as a Scientist in the same lab from Aug. 2003 until May 2005. From Jun. 2005, he took the position of Principal Investigator and set up his own lab in Model Animal Research Institute, Nanjing University.
B
lood vessels arise through two distinct processes, vasculogenesis and angiogenesis. Lymphatic vessels originate from cardinal vein after a subpopulation of blood vascular endothelial cells differentiate into lymphatic endothelial cells, followed by the growth of lymphatic vessels by the process of lymphangiogenesis. Besides their central roles in development and health, both angiogenesis and lymphangiogenesis are also implicated in a variety of diseases such as cancer growth and metastasis.
One of the major goals of our research is to understand the molecular network regulating vascular growth and patterning during development, with particular emphasis on studying the roles of endothelial expressing receptor tyrosine kinases and their ligands in these processes. We have been generating various genetically modified mouse models for analysing molecular circuits in the regulation of endothelial cell differentiation, proliferation and vessel formation. Another major theme of our research is to study molecular mechanisms of tumor metastasis. We are particularly interested in finding out how tumor cells gain access into blood vessels or lymphatic vessels, and how they establish metastatic foci in distant organs. By understanding the underlying mechanisms of tumor vascular development, we aim to develop molecular methods to manipulate these processes and provide novel therapies for future clinical applications. Interesting progress has been made as demonstrated in our recent publications.
Lab members
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Tel.: +86-25-58641512(Office) +86-25-58641522(Lab) Fax: +86-25-58641500 Email: yulong.he@https://www.doczj.com/doc/e51992939.html,
Research Students
? Fei Zhou Luqing Zhang Jun Zhang Bin Shen Liangfa Wang Beibei Lai
Visiting researchers
? Wei Sun Qingxin Meng Fujing Zhang Xin Wang Shijun Zhang
Graduated Students ? Huilian Huang Wencan Han
Technical Staff ? Yanlan Cao
Fig.1 Vegfr3
ΔLBD/ΔLBD mice showed edema due to lack of lymphatic network formation.
Visualization of blood vessels (arrows) and lymphatics in the dorsal skin (dotted white square) by whole-mount immunostaining for PECAM-1 (green) and LYVE-1 (red) in Vegfr3ΔLBD/ΔLBD embryos (E15.5). Note that there is no lymphatic vessel detected in mice homozygous for the LBD deletion and mice show subcutaneous edema (arrowhead). Scale bars, 200 μm.
Fig.2 Fewer endothelial cell numbers in lymphatic capillaries of Akt1/ mice.
A–F: Whole-mount staining for LYVE-1 (green) and PROX1 (red) with ear skins of WT (A, C, and E) and Akt1/ mice (B, D, and F).
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Fig.3 Role and potential mechanism of VEGFR-3 in vascular development.
a. Summary of the vascular phenotypes in the three genetically modified mouse models for VEGFR-3. Loss of the ligand binding or kinase activity of VEGFR-3 affects only lymphangiogenesis but not angiogenesis. Mice null for VEGFR-3 display abnormal angiogenesis and die before the occurrence of lympangiogenesis (indicated in the table as -).
b. Lymph sac (LS) formation. LS formation requires VEGFR-3 kinase activity, but is independent of the direct activation of VEGFR-3 by its cognate ligands (VEGF-C/-D). During embryogenesis (around E10 in mouse), a population of blood vascular endothelial cells (BEC) in the cardinal vein (CV) differentiate into lymphatic endothelial cells (LEC). Then LECs will dissociate from BECs and form lymph sac adjacent to cardinal vein. We have found that VEGFR-3 kinase activity is essential for lymph sac formation as LS does not occur in mice with a kinase inactive VEGFR-3 (Vegfr3TKmut/TKmut ). However, lymph sac formation occurs in the absence of ligand binding activity of VEGFR-3 as shown in Vegfr3ΔLBD/ΔLBD mice. It is possible that VEGFR-3ΔLBD undergoes the ligand independent autophosphorylation as shown in Supplemental Fig. 3. It is also likely that the VEGFR-3 interacting factors, including neuropilin-2 (NRP2, a co-receptor of VEGFR-3), integrin β1 or VEGFR-2, could form complexes with VEGFR-3ΔLBD and activate its kinase activity for downstream signaling. The exact mechanism of lymph sac formation in Vegfr3ΔLBD/ΔLBD mice remains to be investigated.
c. Blood vascular development is independent of VEGFR-3 ligand binding and kinase activity. Shown in this panel is the schematic illustration of the interactions and potential roles of VEGFR-1, VEGFR-2 and VEGFR-3 in the regulation of angiogenesis. VEGFR-1 has been shown to negatively regulate VEGFR-2 signaling by sequestering its ligand VEGF-A. Based on our findings from two genetically modified models, we conclude that VEGFR-3 ligand binding and kinase activity are not required for angiogenesis. At least in cultured endothelial cells (HUVECs), we have found that VEGFR-3 can form heterodimers with VEGFR-2 and regulate its phosphorylation level upon VEGF-A treatment. Therefore, it is possible that VEGFR-3 plays a role in the regulation of blood vessel growth by modulating VEGFR-2 mediated signals. Additionally, as VEGFR-3 can interact with other factors as described above, it is also possible that VEGFR-3 could be activated in a way different from that induced by its cognate ligands, and participates in the regulation of blood vascular development. Further studies are
required to elucidate the mechanism underlying the role of VEGFR-3 in vascular growth.
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chromosome instability and cin-induced diseases
Xingxu Huang Ph.D.
Xingxu Huang received his Ph.D. degree from Nanfang Medical University (Guangzhou, China) in 1998. From 2001 to 2008, Xingxu did his postdoctoral research on roles of cell cycle regulator in development and diseases under the guidance of Dr. Pumin Zhang in Baylor College of Medicine (Houston, Texas, USA). He joined the Faculty of Model Animal Research Center (MARC), Nanjing University in 2008. He is now a professor of genetics and developmental biology and a Principal Investigator in MARC.
T
he inheritance by daughter cells of their whole copy of genome is central to cell proliferation. Fidelity and essence of the chromosomes transmission into two daughter cells is critical for the genome integrity, and is dependent on the mitotic checkpoint. Activation of mitotic checkpoint leads to the final inhibition of Separase, a protease required to dissolve cohesion complex, the sister chromatids linker, to separate sister chromosomes at the metaphase-to-anaphase transition. Therefore, appropriate regulation of Separase is critical to ensure accurate chromosome segregation to prevent chromosome instability (CIN), i.e. abnormal chromosome number, or aneuploidy, a leading cause of spontaneous miscarriages, and a hallmark of many cancers. The manipulation of Separase allows us to explore the molecular biology of chromosome segregation, deregulated Separase-induced CIN, and consequently, the CIN-induced diseases including infertility on germ cells, and tumor on somatic cells.
In vitro experiments demonstrated, under the survey of mitotic checkpoint, Separase is dually controlled by Securin inhibitory binding, and CDK1/CyclinB1 inhibitory phosphorylation major on serine 1126 of human Separase. Using Securin knock-out, Separase knock-out, and Separase dominant phospho-site point mutation mouse embryo stem cell lines, as well as mouse models, we demonstrated that, 1. Securin is not indispensable for Separase inhibition (Mei et al. 2001). 2. The Securin inhibitory binding and inhibitory phosphorylation act redundantly to properly regulate Separase activity (Huang et al. 2005). 3. Separase nonphosphorylatable knock-in resulted in mouse infertility of both sexes by causing completely loss of PGCs, which came from aberrant mitosis, and then CIN on male PGCs (Huang et al. 2008). 4. phosphor-regulatory machinery functions critically for Separase control in early embryogenesis, but not in meiosis (Huang et al. 2009).
Lab members
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Tel.: +86-25-58641517 (Office) +86-25-58641523 (Lab) Fax: +86-25-58641500Email: huangxx@https://www.doczj.com/doc/e51992939.html, xingxuhuang@https://www.doczj.com/doc/e51992939.html,
Further analysis showed, because of the sex specific discrepancy of Securin, deregulation Separase allows a population of female PGCs to survive from chromosome segregation errors and aneuploidy, and run reduced oogenesis. Our results elucidated that Separase phospho-regulation is critical for genome stability in oogenesis, provided the first evidences of pre-zygotic mitotic aneuploidy from Separase deregulation during oogenesis characterized with sexual dimorphism effects (manuscript under revision).
Mitotic checkpoint control of chromosome segregation is achieved by Cdc20 binding with Mad2 to inhibit Securin and Cyclin B1 degradation by APC/C (Anaphase Promoting Complex/cyclosome), then Separase activation. Will and how mitotic checkpoint malfunction facilitate the CIN, then CIN-induced diseases? To address this issue, we created a Cdc20 knockout combined conditional Mad2 binding site mutated Cdc20 mouse model which don’t have mitotic checkpoint. The preliminary data showed neither Cdc20 haploinsufficiency, nor Cdc20 dominant mutation affected gametogenesis, but both mutations promoted tumorigenesis. Interestingly, Cdc20 haploinsufficiency combined with Cdc20 dominant mutation arrested spermatogenesis at spermatocyte stage (Figure 1). Further analyses are being performed.
Technical Assistant ? Jinmei Chen
Graduate Students
? Yinan Du Xinxing Gao Bian Hu Juan Xu
Undergraduate Students ? Zhehao Xu Jiankui Zhou
Trainee in Collaboration ? Juling Feng
Shuai Wang
sections from control and mutant 2-month-old mice.
Fig. 2 Dual regulation of Separase.
Deregulation of Separase leads to chromosome mis-segregation,
then chromosome instability (CIN).
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14
genetic research on bone and joint disease
Qing Jiang Ph.D.
Qing Jiang received his MD degree in Nanjing Medical University in 1989 and PhD degree in Beijing Medical University in 1999. In 2008, he was appointed professor in Nanjing University and moved to Model Animal Research Center as Adjunct Professor for genetic research on bone and joint disease. Now Qing Jiang’s group has established human gene bank of bone and joint disease including osteoarthritis (OA), developmental dysplasia of the hip (DDH), deep venous thrombosis (DVT), ankylosing spondylitis (AS) and osteoporosis (OP).
D
evelopmental dysplasia of the hip (DDH), formerly known as congenital dislocation of the hip, comprises a spectrum of abnormalities, including abnormal acetabular shape (dysplasia) and malposition of the femoral head during embryonic, fetal and infantile growth periods. Most developed countries reported an incidence of 1.5 to 20 cases of DDH per 1000 births, the variation due in part to differences in diagnostic method and timing of evaluation. The incidence in China was estimated about 0.1% to 0.5%. Persistent DDH may result in chronic pain, gait abnormalities and degenerative arthritis. Hereditary factors had been paid more attention to the development of DDH. A genome-wide screening of a Japanese family with acetabular dysplasia identified a linkage on a limited location of the specific chromosome. [ Fig 1]
Our group is focusing on the genetic research of DDH. In the past one year we have discovered TBX4 and ASPN as susceptibility gene of DDH. [Fig 2] Meanwhile we have also excluded DVWA as susceptibility gene of DDH. Now we are still trying to find new susceptibility genes of DDH.
Deep venous thrombosis (DVT) has been observed three months post operatively in 2.4% of patients who have undergone hip arthroplasty (replacement), and in 1.7% of the patients who have undergone knee arthroplasty despite their having received prophylaxis. Symptomatic venous thrombosis occurs after the patients leave hospital, and the risk increases for at least two months after surgery. Thrombosis is a common cause of readmission to hospital subsequent to total hip replacement surgery and constitutes an important health problem in worldwide. It has been suggested that discovery and application of molecular biomarkers that incorporate with deep vein venography could improve the management of patients with DVT. Advances in genomics and proteomics have generated many new candidate markers with potential clinical values. Recently, the discovery of
Lab members
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microRNAs (miRNAs) has opened new avenues for DVT diagnosis and prediction of treatment response. miRNAs are an abundant class of small, nonprotein-coding RNAs that function as negative gene regulators. Because a single miRNA can regulate hundreds of downstream genes with different biologic entities, the information gained from miRNA profiling may be used to detect the onset or progression of DVT. Now our group is trying to investigate the role of serum miRNA in diagnosis and prediction of treatment response of DVT used genome-wide serum miRNA expression analysis.
We have also worked in other common disease related with bone and cartilage. We try to find new susceptibility gene of osteoporosis, adolescent idiopathic scoliosis (AIS) and osteoarthritis (OA). We have found negative results in osteoporosis and adolescent idiopathic scoliosis association research. But we have confirmed new sequence variants in HLA class II/III region are associated with susceptibility to knee osteoarthritis in Chinese Han population (Osteoarthritis & Cartilage 2010). We have also observed significant pathologic changes of Achilles tendon in leptin-deficient mice (Rheumatology International 2010).
The DNA bank for bone and joint disease we had established is still enlarging. Now we have human DNA samples for OA, DDH, DVT, ankylosing spondylitis (AS) and osteoporosis. The tissue bank for cartilage and ligment has been established and enlarged.
Research Associate ? Dongquan Shi
Graduate Students
? Jin Dai Kejie Wang Yanyun Lv Xu Jiang Lisheng Sun Anyun Guo Yong Pang Hongsong Chen Biao Lu Miao Chu Jizheng Qin Wei Sun Juanke Zhu
Technician ? Qiting Ge
Technical Assistant ?
Xueqin Zhu
15
Polymorphism was genotyped using PAGE and DNA size analysis
Fig. 1 Classification of DDH 1.Hip instability, 2.Hip subluxation, 3.Hip dislocation
Fig. 2 ASPN microsatellite polymorphism was genotyped by PAGE and DNA Size analysis.
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cell metabolism and protein prenylation
Chaojun Li Ph.D.
Chao-Jun Li received his Bachelor (1988) and Master (1991) in Zoology, and his Ph.D. in Physiology (1995), all from Nanjing University. He also did his postdoctoral training at the Hong Kong University of Science and Technology from 1996-1998 and Yale University from 1999-2000. He joined Model Animal Research Center of Nanjing University as a professor of Cell Biology in 2008.
1.The mechanism of insulin resistance development.
Insulin can activate two signaling pathways: the PI3K/Akt pathway, which is responsible for glucose metabolism and the MAPK pathway, which is crucial for insulin resistance. But it is unclear exactly how the two pathways coordinate to regulate insulin sensitivity in the pathogenesis of type 2 diabetes mellitus (T2DM). We found that an early response transcription factor Egr-1 could tilt the signaling balance by blocking PI3K/Akt signaling through PTEN and augmenting Erk/MAPK signaling through GGPPS, resulting in insulin resistance in adipocytes. Egr-1, PTEN and GGPPS are upregulated in the fat tissue of T2DM patients and db/db mice. In vivo analysis showed that Egr-1 overexpression in epididymal fat induced systematic insulin resistance in wild-type mice, and loss of Egr-1 function improved whole-body insulin sensitivity in diabetic (db/db) mice. Additionally, Egr-1 inhibition in fat tissue or adipocytes restored IRS-1 tyrosine phosphorylation, upregulated PI3K/Akt, and downregulated Erk/MAPK signaling, and then increased the insulin sensitivity. Moreover, Egr-1 also controlled adipose to release adipokines such as TNF- and IL-6. Therefore, our results reveal a novel pathogenic mechanism of T2DM that Egr-1 can control the balance of PI3K/Akt and MAPK signaling pathways to modulate insulin resistance, which provides a new target for the improvement of insulin resistance 2. Protein prenylation and diseases
The posttranslational modification of proteins by lipids is a key mechanism in the regulation of protein localization and function. Isoprenylation is one of the processes critical for the membrane association of lots of signaling proteins, including G proteins. Geranylgeranyl diphosphate synthase (GGPPS), a key enzyme involved in the isoprenylation process, catalyzes the formation of geranylgeranyl diphosphate (GGPP). GGPP is used to prenylate proteins which have CAAX motif in their carboxyl termini and then
Lab members
Contact Information
Tel.: +86-25-83596289 (Office) Fax: +86-25-83596289 Email: licj@https://www.doczj.com/doc/e51992939.html, licj@https://www.doczj.com/doc/e51992939.html,
the geranylgeranylated proteins can attach to the membrane to support their function. We first identified GGPPS as a directly target gene of Egr-1, which can positively feedback to increase Egr-1 accumulation during chronic stress stimulation through enhance Ras prenylation and membrane association. The sustained accumulation of Egr-1, an acute response transcription factor, is account for chronic disease development when exposed to intermittent low level stress stimulation. To further understand the relationship of protein prenylation and disease. We constructed GGPPS Flp mice and conditionally deleted GGPPS gene in different tissue to examine its functions. We have generated GGPPS knockout mice of cardiac-specific in heart, Sertoli cell specific in testis, hepatocytes specific in liver and adipocyte specific in fat. The detailed function of GGPPS and protein prenylation is examining. The primary data has shown that cardiac-specific GGPPS knockout mice lead to cardiac dilation and severe heart failure, Sertoli cell specific GGPPS knockout mice had the defect of germ cell division and resulted in infertility because of oligospermia or azoospermia.3. Cytokinesis and cancer
The cytokinesis is a dedicated process that a plenty of proteins would interact each other in this time and, more importantly at right places like at contractile ring and central spindle. These proteins orchestrate together to regulate the assembly of late mitosis structures and control mitosis completion. Our interest is focusing the integrity of central spindle structure and the progress of cytokinesis. We will also explore the completion of cytokinesis and cancer progression.
Research Associates ? Xue Bin Feiyan Pan
Graduate Students
? Xiuxing Wang Ning Shen Shan Jiang Na Xu Fan Diao
Shanshan Lai Fanli Meng Yao Ding Jiangnan Wang
17
Fig.1 The model of Egr-1/GGPPS/Erk1/2 pathway regulating insulin resistance in hyperinsulinism.
Hyperinsulinism stress induced Egr-1 accumulation in adipocytes. There are three pathways that Egr-1 can augment insulin resistance:
(1) Egr-1 induces sustained activation of the Erk1/2 MAPK pathway through a GGPPS induced positive feedback of Erk1/2 and Egr-1 activation.
(2) Egr-1 inhibits PI3K/Akt signaling through activating PTEN transcription.
(3) Sustained activation of Erk1/2 can phosphorylate IRS-1 on Ser612 and inhibit its activity.
Fig. 2 Cardiac-specific deletion of GGPPS induced dilated hypertrophy in the mouse.
A. GGPPS knockout mice developed dilated hypertrophy from 4 weeks old.
B. Functional analysis indicated that the the left ventricular volume of GGPPS knockout heart became larger and the cardiac function diminished with ages.
Fig.3 Sertoli cell specific deletion of GGPPS showed a small testis and spermatogenesis defect.
A: the testis size was smaller in GGPPS knock out mice.
B:HE staining for adult mice testes and epididymis showed the spermatogenesis is defect in GGPPS knock out mice.
C: total cell and spermatogonia cell number in tubules decreased but the sertoli cell number had no significant change in GGPPS knock out mice compared to control. t,
testis; e, epididymidis; dd, ductus deferens; sv, seminal vesicles.
18
tumor suppression and mouse tumor models
Geng Liu Ph.D.
Geng Liu received his B.S. degree in Biochemistry from Wuhan University, China and his Ph.D. degree in Gene & Development from University of Texas Graduate School of Biomedical Sciences at Houston in 1999. He continued his postdoctoral training at University of Texas M.D. Anderson Cancer Center under the guidance of Dr. Gigi Lozano where he studied the tumor suppression mechanism of p53 in vivo using genetically engineered mouse models. Dr. Geng Liu joined the Model Animal Research Center of Nanjing University as principal investigator and professor of Genetics in 2006.
F
ound to be mutated in over 50% of human cancers, p53 tumor suppressor is a central player in the defense against aberrant proliferation and malignant transformation. In addition p53 also serves important roles in response to cellular stresses (Figure 1). Numerous upstream signals impinge upon p53 to serve as a checkpoint regulating cell growth and death. Mechanistically, upstream stress signals relieve the inhibition of Mdm2 and Mdm4 on p53. As a transcriptional activator, p53 subsequently turns on the transcription of target genes involving cell cycle control, apoptosis and senescence. Mdm2 is an E3 ubiquitin ligase controlling both p53 stability and transcriptional activity. Mdm4, on the other hand, negatively regulates p53 activity either directly or through collaboration with Mdm2. Previous results suggest Mdm2 and Mdm4 play non-overlapping and synergistic roles in inhibiting p53. Small molecules have also been developed targeting p53-Mdm2/Mdm4 interactions in order to activate p53.
One research focus in our laboratory is to delineate the regulation and functionality of the p53-Mdm2/Mdm4 network during development
Lab members
Graduate Students
? Lai Chen Xueyan He Chenxi Zhang Guoxin Zhang Cheng Shanshan
Technical staff
? Yan Ren Ping Zhou
Contact Information
Tel.: +86-25-58641515 (Office) +86-25-58641519 (Lab) Fax: +86-25-58641500 Email: liugeng@https://www.doczj.com/doc/e51992939.html,
and tumorigenesis. The negative regulations of p53 by Mdm2 and Mdm4 are essential for early mouse embryonic development and tissue homeostasis. Cardiovascular system is the first major system to develop and function in the embryo. We studied p53-Mdm2/Mmdm4 regulation in mouse endothelial lineage and their derivatives. Our results indicate that p53 activation through loss of its negative regulators affects either vascular remodeling or cardiac cushion morphogenesis depending on the nature of the inhibitor and gene dose (Figure 2). Congenital heart defects (CHD) are the most prevalent birth defects in humans influenced by genetic susceptibility and maternal exposure during fetal development. Our work also provided new evidence in model systems to suggest a possible molecular link between risk factors and the development of congenital heart defect. We have also made progress in studying the regulation of p53 at other levels by establishing a BAC transgenic reporter system (Figure 3).