Publications by authors named "Huashun Li"

29 Publications

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Interference of commissural connections through the genu of the corpus callosum specifically impairs sensorimotor gating.

Behav Brain Res 2021 May 25;411:113383. Epub 2021 May 25.

Key Lab of Organ Development and Regeneration of Zhejiang Province, Institute of Life Sciences, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, 311121, China. Electronic address:

White matter abnormalities in schizophrenic patients are characterized as regional tract-specific. Myelin loss at the genu of the corpus callosum (GCC) is one of the most consistent findings in schizophrenic patients across the different populations. We characterized the axons that pass through the GCC by stereotactically injecting an anterograde axonal tracing viral vector into the forceps minor of the corpus callosum in one hemisphere, and identified the homotopic brain structures that have commissural connections in the two hemispheres of the prefrontal cortex, including the anterior cingulate area, the prelimbic area, the secondary motor area, and the dorsal part of the agranular insular area, along with commissural connections with the primary motor area, caudoputamen, and claustrum. To investigate whether dysmyelination in these commissural connections is critical for the development of schizophrenia symptoms, we generated a mouse model with focal demyelination at the GCC by stereotactically injecting demyelinating agent lysolecithin into this site, and tested these mice in a battery of behavioral tasks that are used to model the schizophrenia-like symptom domains. We found that demyelination at the GCC influenced neither the social interest or mood state, nor the locomotive activity or motor coordination. Nevertheless, it specifically reduced the prepulse inhibition of acoustic startle that is a well-known measure of sensorimotor gating. This study advances our understanding of the pathophysiological contributions of the GCC-specific white matter lesion to the related disease, and demonstrates an indispensable role of interhemispheric communication between the frontal cortices for the top-down regulation of the sensorimotor gating.
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http://dx.doi.org/10.1016/j.bbr.2021.113383DOI Listing
May 2021

Robo1-specific chimeric antigen receptor natural killer cell therapy for pancreatic ductal adenocarcinoma with liver metastasis.

J Cancer Res Ther 2020;16(2):393-396

Department of Interventional Radiology; Department of Radiology, Luwan Branch, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.

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http://dx.doi.org/10.4103/jcrt.JCRT_190_20DOI Listing
September 2020

Oocyte-specific deletion of Gα induces oxidative stress and deteriorates oocyte quality in mice.

Exp Cell Res 2018 09 17;370(2):579-590. Epub 2018 Jul 17.

School of Life Science and Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan 250100, PR China. Electronic address:

The stimulatory heterotrimeric G protein alpha subunit (Gα) is a ubiquitous guanine nucleotide-binding protein that regulates the intracellular cAMP signaling pathway and consequently participates in a wide range of biological events. In the reproductive system, despite Gα being associated with oocyte meiotic arrest in vitro, the exact role of Gα in female fertility in vivo remains largely unknown. Here, we generated oocyte-specific Gα knockout mice by using the Cre/LoxP system. We observed that the deletion of Gα caused complete female infertility. Exclusion of post-implantation abnormalities, oogenesis, fertilization, and early embryo development was subsequently monitored; meiosis in Gα-deficient oocytes precociously resumed in only 43% of antral follicles from mutant mice, indicating that alteration of meiotic pause was not the key factor in infertility. Ovulation process and number were normal, but the rate of morphological abnormal oocytes was apparently increased; spindle organization, fertilization, and early embryo development were impaired. Furthermore, the level of ROS (reactive oxygen species) and the mitochondrial aggregation increased, and antioxidant glutathione (GSH) content, ATP level, mtDNA copy number, and mitochondrial membrane potential decreased in Gα-deficient oocytes. GV oocytes from mutant mice showed early-stage apoptosis. Meanwhile, the Gα knockout-induced decline in oocyte quality and low developmental potential was partially rescued by antioxidant supplementation. To sum up, our results are the first to reveal that the profile of Gα oocyte-specific deletion caused female infertility in vivo, and oxidative stress plays an important role in this event.
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http://dx.doi.org/10.1016/j.yexcr.2018.07.023DOI Listing
September 2018

Targeting Polo-like Kinase 1 by a Novel Pyrrole-Imidazole Polyamide-Hoechst Conjugate Suppresses Tumor Growth .

Mol Cancer Ther 2018 05 26;17(5):988-1002. Epub 2018 Feb 26.

Shenzhen Key Laboratory for Molecular Biology of Neural Development, Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong, China.

The serine/threonine kinase Polo-like kinase 1 (Plk1) plays a pivotal role in cell proliferation and has been validated as a promising anticancer drug target. However, very limited success has been achieved in clinical applications using existing Plk1 inhibitors, due to lack of sufficient specificity toward Plk1. To develop a novel Plk1 inhibitor with high selectivity and efficacy, we designed and synthesized a pyrrole-imidazole polyamide-Hoechst conjugate, PIP3, targeted to specific DNA sequence in the promoter. PIP3 could specifically inhibit the cell cycle-regulated Plk1 expression and consequently retard tumor cell growth. Cancer cells treated with PIP3 exhibited severe mitotic defects and increased apoptosis, whereas normal cells were not affected by PIP3 treatment. Furthermore, subcutaneous injection of PIP3 into mice bearing human cancer xenografts induced significant tumor growth suppression with low host toxicity. Therefore, PIP3 exhibits the potential as an effective agent for targeted cancer therapy. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0747DOI Listing
May 2018

Mammalian Numb protein antagonizes Notch by controlling postendocytic trafficking of the Notch ligand Delta-like 4.

J Biol Chem 2017 12 17;292(50):20628-20643. Epub 2017 Oct 17.

From the Shenzhen Key Laboratory for Molecular Biology of Neural Development, Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China,

The biological antagonism between the signaling proteins Numb and Notch has been implicated in the regulation of many developmental processes, especially in asymmetric cell division. Mechanistic studies show that Numb inactivates Notch via endocytosis and proteasomal degradation that directly reduce Notch protein levels at the cell surface. However, some aspects of how Numb antagonizes Notch remain unclear. Here, we report a novel mechanism in which Numb acts as a Notch antagonist by controlling the intracellular destination and stability of the Notch ligand Delta-like 4 (Dll4) through a postendocytic-sorting process. We observed that Numb/Numblike knockdown increases the stability and cell-surface accumulation of Dll4. Further study indicated that Numb acts as a sorting switch to control the postendocytic trafficking of Dll4. Of note, the Numb/Numblike knockdown decreased Dll4 delivery to the lysosome, while increasing the recycling of Dll4 to the plasma membrane. Moreover, we demonstrate that this enrichment of Dll4 at the cell surface within Numb/Numblike knockdown cells could activate Notch signaling in neighboring cells. We also provide evidence that Numb negatively controls the Dll4 plasma membrane recycling through a well-documented recycling regulator protein AP1. In conclusion, our study has uncovered a molecular mechanism whereby Numb regulates the endocytic trafficking of the Notch ligand Dll4. Our findings provide a new perspective on how Numb counteracts Notch signaling and sheds additional critical insights into the antagonistic relationship between Numb and Notch signaling.
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http://dx.doi.org/10.1074/jbc.M117.800946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733598PMC
December 2017

Numb positively regulates autophagic flux via regulating lysosomal function.

Biochem Biophys Res Commun 2017 09 15;491(3):780-786. Epub 2017 Jul 15.

Shenzhen Key Laboratory for Molecular Biology of Neural Development, Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China. Electronic address:

Autophagy is a lysosome-dependent catabolic process involving in the degradation and recycling of unnecessary or damaged proteins and organelles. Emerging evidence indicates that autophagy dysfunction is closely related to various human diseases including cancer, aging, myopathies and neurodegenerative disorders. Here, using genetic knockdown, we uncover the role of Numb, an endocytic adaptor protein, in regulating the late steps of autophagy. We found that Numb depletion led to the accumulation of autophagic vacuole, as verified by RFP-LC3 staining combined with transmission electron microscopy. Further investigation indicated that Numb depletion impaired autophagic degradation through inhibiting the activities of lysosomal enzymes (Cathepsin D, β-glucuronidase and β-glucosidase). Moreover, Numb depletion induced elevation of lysosomal pH values and decrease of glycosylated lysosome-associated membrane proteins. We further observed that Rab7 activity was inhibited in Numb-depleted cells. Together, our findings revealed a novel function of Numb and its likely mechanism in regulation of autophagy events.
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http://dx.doi.org/10.1016/j.bbrc.2017.07.084DOI Listing
September 2017

Deletion of Numb/Numblike in glutamatergic neurons leads to anxiety-like behavior in mice.

Brain Res 2017 06 24;1665:36-49. Epub 2017 Mar 24.

SARITEX Center for Stem Cell Engineering Translational Medicine, Shanghai East Hospital, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200123, China; Nerdbio Inc., SIP Biobay, Jiangsu 215213, China. Electronic address:

Endocytic adaptor protein Numb is the first identified cell fate determinant in Drosophila melanogaster. It has been implicated in Notch signaling pathway and regulation of neural stem cells proliferation in the central nervous system. Numb is also expressed in postmitotic neurons, in vitro studies showed that Numb is involved in neuronal morphologic development, such as neurite growth, axonal growth and spine development. However, in vivo functions of Numb in the postmitotic neurons are largely unknown. Here we show that deletion of Numb/Numblike in glutamatergic neurons causes anxiety-like behavior in mouse. In this study, we conditionally deleted Numb and its homologous gene Numblike in the glutamatergic neurons in dorsal forebrain, and thoroughly characterized the behavioral phenotypes of mutant mice. On a battery of tests for anxiety-like behavior, the conditional double knockout mice showed increased anxiety-like behavior on light/dark exploration and novel open field tests, but not on elevated zero maze tests. The conditional double knockout mice also displayed novelty induced hyperactivity in novel open field test. Control measures of general health, motor functions, startle response, sensorimotor gating, depression-related behaviors did not show differences between genotypes. Our present findings provide new insight into the indispensable functions of Numb/Numblike in the brain and behavior, and suggest that Numb/Numblike may play a role in mediating neuronal functions that underlie behaviors related to anxiety.
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http://dx.doi.org/10.1016/j.brainres.2017.02.025DOI Listing
June 2017

Heterotrimeric G Stimulatory Protein α Subunit Is Required for Intestinal Smooth Muscle Contraction in Mice.

Gastroenterology 2017 04 30;152(5):1114-1125.e5. Epub 2016 Dec 30.

The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Qilu Hospital of Shandong University, Jinan, China. Electronic address:

Background & Aims: The α subunit of the heterotrimeric G stimulatory protein (Gsa), encoded by the guanine nucleotide binding protein, α-stimulating gene (Gnas, in mice), is expressed ubiquitously and mediates receptor-stimulated production of cyclic adenosine monophosphate and activation of the protein kinase A signaling pathway. We investigated the roles of Gsa in vivo in smooth muscle cells of mice.

Methods: We performed studies of mice with Cre recombinase-mediated disruption of Gnas in smooth muscle cells (Gsa and SM22-CreER, induced in adult mice by tamoxifen). Intestinal tissues were collected for histologic, biochemical, molecular, cell biology, and physiology analyses. Intestinal function was assessed in mice using the whole-gut transit time test. We compared gene expression patterns of intestinal smooth muscle from mice with vs without disruption of Gnas. Biopsy specimens from ileum of patients with chronic intestinal pseudo-obstruction and age-matched control biopsies were analyzed by immunohistochemistry.

Results: Disruption of Gnas in smooth muscle of mice reduced intestinal motility and led to death within 4 weeks. Tamoxifen-induced disruption of Gnas in adult mice impaired contraction of intestinal smooth muscle and peristalsis. More than 80% of these died within 3 months of tamoxifen exposure, with features of intestinal pseudo-obstruction characterized by chronic intestinal dilation and dysmotility. Gsa deficiency reduced intestinal levels of cyclic adenosine monophosphate and transcriptional activity of the cyclic adenosine monophosphate response element binding protein 1 (CREB1); this resulted in decreased expression of the forkhead box F1 gene (Foxf1) and protein, and contractile proteins, such as myosin heavy chain 11; actin, α2, smooth muscle, aorta; calponin 1; and myosin light chain kinase. We found decreased levels of Gsa, FOXF1, CREB1, and phosphorylated CREB1 proteins in intestinal muscle layers of patients with chronic intestinal pseudo-obstruction, compared with tissues from controls.

Conclusions: Gsa is required for intestinal smooth muscle contraction in mice, and its levels are reduced in ileum biopsies of patients with chronic intestinal pseudo-obstruction. Mice with disruption of Gnas might be used to study human chronic intestinal pseudo-obstruction.
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http://dx.doi.org/10.1053/j.gastro.2016.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430528PMC
April 2017

Lgl1 Is Required for Olfaction and Development of Olfactory Bulb in Mice.

PLoS One 2016 7;11(9):e0162126. Epub 2016 Sep 7.

Institute of Developmental Biology, School of Life Science, Shandong University, Jinan, 250100, Shandong, China.

Lethal giant larvae 1 (Lgl1) was initially identified as a tumor suppressor in Drosophila and functioned as a key regulator of epithelial polarity and asymmetric cell division. In this study, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in olfactory bulb (OB). Next, we examined the effects of Lgl1 loss in the OB. First, we determined the expression patterns of Lgl1 in the neurogenic regions of the embryonic dorsal region of the LGE (dLGE) and postnatal OB. Furthermore, the Lgl1 conditional mutants exhibited abnormal morphological characteristics of the OB. Our behavioral analysis exhibited greatly impaired olfaction in Lgl1 mutant mice. To elucidate the possible mechanisms of impaired olfaction in Lgl1 mutant mice, we investigated the development of the OB. Interestingly, reduced thickness of the MCL and decreased density of mitral cells (MCs) were observed in Lgl1 mutant mice. Additionally, we observed a dramatic loss in SP8+ interneurons (e.g. calretinin and GABAergic/non-dopaminergic interneurons) in the GL of the OB. Our results demonstrate that Lgl1 is required for the development of the OB and the deletion of Lgl1 results in impaired olfaction in mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0162126PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014313PMC
August 2017

NUMB negatively regulates the epithelial-mesenchymal transition of triple-negative breast cancer by antagonizing Notch signaling.

Oncotarget 2016 Sep;7(38):61036-61053

Shenzhen Key Laboratory for Molecular Biology of Neural Development, Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China.

Triple-negative breast cancer (TNBC), an aggressive subtype of breast cancer with higher rates of early relapse and metastasis, is frequently associated with aberrant activation of epithelial-mesenchymal transition (EMT). Nonetheless, how EMT is initiated and regulated during TNBC progression is not well understood. Here, we report that NUMB is a negative regulator of EMT in both human mammary epithelial cells and breast cancer cells. Reduced NUMB expression was significantly associated with elevated EMT in TNBC. Conversely, overexpression of NUMB strongly attenuated the EMT program and metastasis of TNBC cell lines. Interestingly, we showed that NUMB employs different molecular mechanisms to regulate EMT. In normal mammary epithelial cells and breast cancer cells expressing wild-type p53, NUMB suppressed EMT by stabilizing p53. However, in TNBC cells, loss of NUMB facilitated the EMT program by activating Notch signaling. Consistent with these findings, low NUMB expression and high Notch activity were significantly correlated with the TNBC subtype in patients. Collectively, these findings reveal novel molecular mechanisms of NUMB in the regulation of breast tumor EMT, especially in TNBC.
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http://dx.doi.org/10.18632/oncotarget.11062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5308634PMC
September 2016

Numb regulates vesicular docking for homotypic fusion of early endosomes via membrane recruitment of Mon1b.

Cell Res 2016 05 18;26(5):593-612. Epub 2016 Mar 18.

SARITEX Center for Stem Cell Engineering Translational Medicine, Shanghai East Hospital, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200123, China.

Numb is an endocytic protein that plays crucial roles in diverse cellular processes such as asymmetric cell division, cell migration and differentiation. However, the molecular mechanism by which Numb regulates endocytic trafficking is poorly understood. Here, we demonstrate that Numb is a docking regulator for homotypic fusion of early endosomes (EEs). Numb depletion causes clustered but unfused EEs, which can be rescued by overexpressing cytosolic Numb 65 and Numb 71 but not plasma membrane-attached Numb 66 or Numb 72. Time-lapse analysis reveals that paired vesicles tend to tether but not fuse with each other in the absence of Numb. We further show that Numb binds to another docking regulator, Mon1b, and is required for the recruitment of cytosolic Mon1b to the EE membrane. Consistent with this, deletion of Mon1b causes similar defects in EE fusion. Our study thus identifies a novel mechanism by which Numb regulates endocytic sorting by mediating EE fusion.
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http://dx.doi.org/10.1038/cr.2016.34DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856763PMC
May 2016

Transferrin Receptor Controls AMPA Receptor Trafficking Efficiency and Synaptic Plasticity.

Sci Rep 2016 Feb 16;6:21019. Epub 2016 Feb 16.

SARITEX Center for Stem Cell Engineering Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine &Advanced Institute of Translational Medicine, Shanghai 200123, China.

Transferrin receptor (TFR) is an important iron transporter regulating iron homeostasis and has long been used as a marker for clathrin mediated endocytosis. However, little is known about its additional function other than iron transport in the development of central nervous system (CNS). Here we demonstrate that TFR functions as a regulator to control AMPA receptor trafficking efficiency and synaptic plasticity. The conditional knockout (KO) of TFR in neural progenitor cells causes mice to develop progressive epileptic seizure, and dramatically reduces basal synaptic transmission and long-term potentiation (LTP). We further demonstrate that TFR KO remarkably reduces the binding efficiency of GluR2 to AP2 and subsequently decreases AMPA receptor endocytosis and recycling. Thus, our study reveals that TFR functions as a novel regulator to control AMPA trafficking efficiency and synaptic plasticity.
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http://dx.doi.org/10.1038/srep21019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754636PMC
February 2016

G-Protein α-Subunit Gsα Is Required for Craniofacial Morphogenesis.

PLoS One 2016 9;11(2):e0147535. Epub 2016 Feb 9.

West China Developmental & Stem Cell Institute, West China Second Hospital, and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, China.

The heterotrimeric G protein subunit Gsα couples receptors to activate adenylyl cyclase and is required for the intracellular cAMP response and protein kinase A (PKA) activation. Gsα is ubiquitously expressed in many cell types; however, the role of Gsα in neural crest cells (NCCs) remains unclear. Here we report that NCCs-specific Gsα knockout mice die within hours after birth and exhibit dramatic craniofacial malformations, including hypoplastic maxilla and mandible, cleft palate and craniofacial skeleton defects. Histological and anatomical analysis reveal that the cleft palate in Gsα knockout mice is a secondary defect resulting from craniofacial skeleton deficiencies. In Gsα knockout mice, the morphologies of NCCs-derived cranial nerves are normal, but the development of dorsal root and sympathetic ganglia are impaired. Furthermore, loss of Gsα in NCCs does not affect cranial NCCs migration or cell proliferation, but significantly accelerate osteochondrogenic differentiation. Taken together, our study suggests that Gsα is required for neural crest cells-derived craniofacial development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147535PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747491PMC
July 2016

Gαs Relays Sphingosine-1-Phosphate Receptor 1 Signaling to Stabilize Vascular Endothelial-Cadherin at Endothelial Junctions to Control Mouse Embryonic Vascular Integrity.

J Genet Genomics 2015 Nov 20;42(11):613-624. Epub 2015 Oct 20.

West China Developmental and Stem Cell Institute, West China Second Hospital, Sichuan University, Chengdu 610041, China; State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu 610041, China; SARITEX Center for Stem Cell Engineering Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200123, China; Nerdbio Inc., SIP Biobay, Suzhou 215213, China. Electronic address:

Sphingosine-1-phosphate receptor 1 (S1PR1), a G protein-coupled receptor (GPCR), controls vascular stability by stabilizing vascular endothelial (VE)-cadherin junctional localization and inhibiting vascular endothelial growth factor receptor 2 (VEGFR2) signaling. However, the molecular mechanisms that link S1PR1 signaling to intracellular effectors remain unknown. In this study, we demonstrate that the heterotrimeric G protein subfamily member Gαs, encoded by GNAS, acts as a relay mediator of S1PR1 signaling to control vascular integrity by stabilizing VE-cadherin at endothelial junctions. The endothelial cell-specific deletion of Gαs in mice causes early embryonic lethality with massive hemorrhage and a disorganized vasculature. The immunostaining results revealed that Gαs deletion remarkably reduces the junctional localization of VE-cadherin, whereas the mural cell coverage of the vessels is not impaired. In addition, we found that Gαs depletion blocks the S1PR1-activation induced VE-cadherin stabilization at junctions, supporting that Gαs acts downstream of S1PR1 signaling. Thus, our results demonstrate that Gαs is an essential mediator to relay S1PR1 signaling and maintain vascular integrity.
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http://dx.doi.org/10.1016/j.jgg.2015.08.006DOI Listing
November 2015

Wnt3a suppresses Wnt/β-catenin signaling and cancer cell proliferation following serum deprivation.

Exp Cell Res 2016 Feb 28;341(1):32-41. Epub 2015 Nov 28.

Key Laboratory of Arrhythmias of the Ministry of Education of China, Tongji University School of Medicine, Shanghai 200092, China; Research Center for Translational Medicine, East Hospital, Tongji University School of Medicine, Shanghai 200120, China. Electronic address:

Canonical Wnt/β-catenin signaling is often aberrantly activated in tumor cells and required for tumor growth. The internalization of Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) induced by Wnt ligands is commonly thought to be critical for Wnt/β-catenin signaling activation. However, in contrast to theses previous studies, we here show that persistent excessive stimulation with a canonical Wnt ligand Wnt3a could induce a long-term decreased expression level of membrane LRP6, which prevented nuclear β-catenin accumulation and tumor cell;proliferation. Importantly, Wnt3a was robustly upregulated following serum deprivation. The upregulated Wnt3a under serum deprivation was responsible for LRP6 internalization, decreased accumulation of nuclear β-catenin, and further inhibition of tumor cell proliferation. It has well been known that insufficient blood supply during tumor development occurs frequently, causing a worsening environment for tumor growth. Therefore, these results reveal a novel inhibitory role of Wnt3a on canonical Wnt/β-catenin signaling and cancer cell proliferation when there is an insufficient blood supply during tumor development, which might be a potential mechanism for tumor evasion within a worsening environment.
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http://dx.doi.org/10.1016/j.yexcr.2015.11.025DOI Listing
February 2016

LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration.

Sci Rep 2015 Nov 9;5:16232. Epub 2015 Nov 9.

School of Life Science and Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan 250100, China.

The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1(Atoh1) CKO) to investigate the function of LKB1 in cerebellar development. The LKB1(Atoh1) CKO mice displayed motor dysfunction. In the LKB1(Atoh1) CKO cerebellum, the overall structure had a larger volume and more lobules. LKB1 inactivation led to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1(Atoh1) CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development.
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http://dx.doi.org/10.1038/srep16232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4637891PMC
November 2015

LKB1 Is Required for the Development and Maintenance of Stereocilia in Inner Ear Hair Cells in Mice.

PLoS One 2015 14;10(8):e0135841. Epub 2015 Aug 14.

Institute of Developmental Biology, School of Life Science, Shandong University, Jinan, Shandong, China.

The LKB1 gene, which encodes a serine/threonine kinase, was discovered to play crucial roles in cell differentiation, proliferation, and the establishment of cell polarity. In our study, LKB1 conditional knockout mice (Atoh1-LKB1-/- mice) were generated to investigate LKB1 function in the inner ear. Tests of auditory brainstem response and distortion product otoacoustic emissions revealed significant decreases in the hearing sensitivities of the Atoh1-LKB1-/- mice. In Atoh1-LKB1-/- mice, malformations of hair cell stereocilliary bundles were present as early as postnatal day 1 (P1), a time long before the maturation of the hair cell bundles. In addition, we also observed outer hair cell (OHC) loss starting at P14. The impaired stereocilliary bundles occurred long before the presence of hair cell loss. Stereociliary cytoskeletal structure depends on the core actin-based cytoskeleton and several actin-binding proteins. By Western blot, we examined actin-binding proteins, specifically ERM (ezrin/radixin/moesin) proteins involved in the regulation of the actin cytoskeleton of hair cell stereocilia. Our results revealed that the phosphorylation of ERM proteins (pERM) was significantly decreased in mutant mice. Thus, we propose that the decreased pERM may be a key factor for the impaired stereocillia function, and the damaged stereocillia may induce hair cell loss and hearing impairments. Taken together, our data indicates that LKB1 is required for the development and maintenance of stereocilia in the inner ear.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135841PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537123PMC
May 2016

Downregulation of Glutamate Transporter EAAT4 by Conditional Knockout of Rheb1 in Cerebellar Purkinje Cells.

Cerebellum 2016 06;15(3):314-21

Department of Neurobiology, Zhejiang University School of Medicine, 866 Yu Hang Tang Road, Hangzhou, 310058, China.

Excitatory amino acid transporter 4 (EAAT4) is believed to be critical to the synaptic activity of cerebellar Purkinje cells by limiting extracellular glutamate concentrations and facilitating the induction of long-term depression. However, the modulation of EAAT4 expression has not been elucidated. It has been shown that Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) signaling plays essential roles in the regulation of protein translation, cell size, and cell growth. In addition, we previously found that a cascade including mTOR suppression and Akt activation induces increased expression of EAAT2 in astrocytes. In the present work, we explored whether Rheb/mTOR signaling is involved in the regulation of EAAT4 expression using conditional Rheb1 knockout mice. Our results demonstrated that Rheb1 deficiency resulted in the downregulation of EAAT4 expression, as well as decreased activity of mTOR and increased activity of Akt. The downregulation of EAAT4 was also confirmed by reduced EAAT4 currents and slowed kinetics of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor-mediated currents. On the other hand, conditional knockout of Rheb1 did not alter the morphology of Purkinje cell layer and the number of Purkinje cells. Overall, our findings suggest that small GTPase Rheb1 is a modulator in the expression of EAAT4 in Purkinje cells.
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http://dx.doi.org/10.1007/s12311-015-0701-9DOI Listing
June 2016

Central nervous system-specific knockout of Brg1 causes growth retardation and neuronal degeneration.

Brain Res 2015 Oct 30;1622:186-95. Epub 2015 Jun 30.

West China Institute of Women and Children׳s Health, West China Second University Hospital, Sichuan University, China; Key Laboratory of Obstetric & Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, China. Electronic address:

Changes in chromatin structure (chromatin remodeling) are essential regulatory processes for neuronal development, but the molecular mechanisms are unclear. The aim of the present study was to assess the effects of conditional knockout (Ko) of the Brahma-related gene-1 (Brg1) in the mouse central nervous system (CNS) on postnatal development. Brg1 was deleted in the CNS by crossing mice carrying the Brg1 conditional allele with a transgenic line expressing Cre under the control of the Nex 1 promoter. Brg1, PSD-95, NR2A and NR2B protein expressions were assessed using western blotting. Immunofluorescence, Nissl and TUNEL staining were used to assess cortical neuron viability. Hippocampal neurons were extracted from mouse embryos to observe the effects of neuronal degeneration associated with oxidative stress using Paraquat dichloride x-hydrate or 80% oxygen. Brg1(fx/fx);NEX-Cre mice were significantly smaller in both body size and brain size after P35 conditional Ko of Brg1 in mouse cortical progenitors. The amount of neurons and their dendritic branches were significantly reduced in Brg1 Ko cortexes during early postnatal development. Absence of Brg1 may result in increased number of astrocytes. Loss of Brg1 increased damaged and dying neurons associated with oxidative stress. Furthermore, loss of NR2A in the Brg1 Ko cortex during early postnatal development, and delayed the NR2B-NR2A switch. Therefore, Brg1 may play a critical role in neuronal growth by regulating the NR2B-NR2A switch. Our findings provide an insight in chromatin remodeling regulation in postnatal neuronal development.
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http://dx.doi.org/10.1016/j.brainres.2015.06.027DOI Listing
October 2015

Gαs regulates asymmetric cell division of cortical progenitors by controlling Numb mediated Notch signaling suppression.

Neurosci Lett 2015 Jun 24;597:97-103. Epub 2015 Apr 24.

West China Developmental and Stem Cell Institute, West China Second Hospital, and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China; Shenzhen Key Laboratory for Molecular Biology of Neural Development, Laboratory of Developmental and Regenerative biology, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China; SARITEX Center for Stem Cell Engineering Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200123, China; Nerdbio Inc. SIP Biobay, Jiangsu 215213, China. Electronic address:

Asymmetric cell division, which plays fundamental roles in generating cell diversity during development, requires elaborate interactions between extrinsic cues and intrinsic cues. However, the precise nature of this type of interaction and its involving signaling mechanisms are poorly understood. Here, we demonstrate that Gαs is present in the proliferative region of ventricular zone in mouse developing neocortex and co-localizes with intrinsic cell fate determinant protein Numb in dividing apical progenitors. Targeted ablation of Gαs subunit in the cortical progenitor causes an alteration from asymmetric to symmetric cell division, consequently leading to increased progenitor proliferation. Mechanistically, we show that Gαs deletion significantly reduces Numb expression and activates notch signaling. Therefore, these results reveal a novel role of Gαs in control of neural progenitor asymmetric cell division via suppressing Numb mediated Notch signaling inhibition.
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http://dx.doi.org/10.1016/j.neulet.2015.04.034DOI Listing
June 2015

Role of Numb expression and nuclear translocation in endometrial cancer.

Oncol Lett 2015 Apr 27;9(4):1531-1536. Epub 2015 Jan 27.

Department of Obstetrics and Gynecology, West China Second Hospital, Sichuan University, Chengdu, Sichuan, P.R. China ; Department of Obstetrics and Gynecology, Second Affiliated Hospital of Chongqing Medical University, Chongqing, P.R. China.

The cell fate determinant Numb orchestrates numerous cell physiological and pathological processes and previous evidence has indicated that Numb expression is associated with tumorigenesis. The present study investigated the potential role of Numb in endometrial cancer (EC). Numb expression was compared between the normal endometrium and EC tissue by immunohistochemistry, and the protein levels were assessed by western blotting and confocal microscopy in the human endometrial HEC-1B cancer cell line and normal epithelial cells. The intracellular localization of Numb in HEC-1B cells was examined by immunofluorescence. Numb was found to be expressed at higher levels in endometrial cancer compared with the normal endometrium. Although Numb localizes to the cytoplasm and plasma membrane in the normal epithelium, the present study found that Numb accumulated in the nucleus of HEC-1B cells. The present data reveals the upregulation of Numb expression in EC tissues and indicates that Numb plays a role in the occurrence of EC, which may be mediated by its translocation into the nucleus. The role of Numb in cancer development requires additional investigation.
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http://dx.doi.org/10.3892/ol.2015.2901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4356328PMC
April 2015

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice.

Dev Biol 2014 Nov 19;395(1):167-81. Epub 2014 Jul 19.

Key Laboratory of the Ministry of Education for Experimental Teratology and School of Life Science, Shandong University, Jinan 250100, China. Electronic address:

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs.
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http://dx.doi.org/10.1016/j.ydbio.2014.07.007DOI Listing
November 2014

PTEN regulation of the proliferation and differentiation of auditory progenitors through the PTEN/PI3K/Akt-signaling pathway in mice.

Neuroreport 2014 Feb;25(3):177-83

aKey Laboratory of the Ministry of Education for Experimental Teratology and School of Life Science, Shandong University, Jinan bSARITEX Center for Stem Cell Engineering Translational Medicine, Shanghai East Hospital cAdvanced Institute of Translational Medicine, Tongji University School of Medicine dCenter for Stem Cell and Nano-Medicine, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, China.

The organ of Corti, which is the sensory organ of hearing, consists of a single row of inner hair cells and three rows of outer hair cells in mice. The auditory hair cells develop from auditory progenitors. Hair cell development is related to several genes, including PTEN. Homozygous null mutant (PTEN(-/-)) mice die at around embryonic day 9, when hair cells are extremely immature. Moreover, in heterozygous PTEN knockout mice, it was found that PTEN regulates the proliferation of auditory progenitors. However, little is known about the molecular mechanism underlying this regulation. In the present study, we generated PTEN conditional knockout in the inner ear of mice and studied the aforementioned molecular mechanisms. Our results showed that PTEN knockout resulted in supernumerary hair cells, increased p-Akt level, and decreased p27(kip1) level. Furthermore, the presence of supernumerary hair cells could be explained by the delayed withdrawal of auditory progenitors from the cell cycle. The increased p-Akt level correlates with p27(kip1) downregulation in the cochlea in the Pax2-PTEN mice. The reduced p27(kip1) could not maintain the auditory progenitors in the nonproliferative state and some progenitors continued to divide. Consequently, additional progenitors differentiated into supernumerary hair cells. We suggest that PTEN regulates p27(kip1) through p-Akt, thereby regulating the proliferation and differentiation of auditory progenitors.
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http://dx.doi.org/10.1097/WNR.0000000000000069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906289PMC
February 2014

POSH localizes activated Rac1 to control the formation of cytoplasmic dilation of the leading process and neuronal migration.

Cell Rep 2012 Sep 6;2(3):640-51. Epub 2012 Sep 6.

State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

The formation of proximal cytoplasmic dilation in the leading process (PCDLP) of migratory neocortical neurons is crucial for somal translocation and neuronal migration, processes that require the elaborate coordination of F-actin dynamics, centrosomal movement, and nucleokinesis. However, the underlying molecular mechanisms remain poorly understood. Here, we show that the Rac1-interacting scaffold protein POSH is essential for neuronal migration in vivo. We demonstrate that POSH is concentrated in the PCDLP and that knockdown of POSH impairs PCDLP formation, centrosome translocation, and nucleokinesis. Furthermore, POSH colocalizes with F-actin and the activated form of Rac1. Knockdown of POSH impairs F-actin assembly and delocalizes activated Rac1. Interference of Rac1 activity also disrupts F-actin assembly and PCDLP formation and perturbs neuronal migration. Thus, we have uncovered a mechanism by which POSH regulates the localization of activated Rac1 and F-actin assembly to control PCDLP formation and subsequent somal translocation of migratory neurons.
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http://dx.doi.org/10.1016/j.celrep.2012.08.007DOI Listing
September 2012

Immune-related GTPase M (IRGM1) regulates neuronal autophagy in a mouse model of stroke.

Autophagy 2012 Nov 9;8(11):1621-7. Epub 2012 Aug 9.

Department of Immunology, Infection and Immunity, Key Laboratory of Heilongjiang Province, Harbin Medical University, Harbin, China.

Autophagy is an important cellular recycling mechanism through self-digestion in responses to cellular stress such as starvation. Studies have shown that autophagy is involved in maintaining the homeostasis of the neural system during stroke. However, molecular mechanisms underlying neuronal autophagy in ischemic stroke remain poorly understood. Previously, we and others have shown that immune-related GTPase M (IRGM; termed IRGM1 in the mouse nomenclature) can regulate the survival of immune cells through autophagy in response to infections and autoimmune conditions. Here, using a permanent middle cerebral artery occlusion (pMCAO) mouse model, we found that IRGM1 was upregulated in the ischemic side of the brain, which was accompanied by a significant autophagic response. In contrast, neuronal autophagy was almost complete lost in Irgm1 knockout (KO) mice after pMCAO induction. In addition, the infarct volume in the Irgm1-KO pMCAO mice was significantly increased as compared to wild-type mice. Histological studies suggested that, at the early stage (within 24 h) of ischemia, the IRGM1-dependent autophagic response is associated with a protection of neurons from necrosis in the ischemic core but a promotion of neuronal apoptosis in the penumbra area. These data demonstrate a novel role of IRGM1 in regulating neuronal autophagy and survival during ischemic stroke.
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http://dx.doi.org/10.4161/auto.21561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494591PMC
November 2012

Immune-related GTPase Irgm1 exacerbates experimental auto-immune encephalomyelitis by promoting the disruption of blood-brain barrier and blood-cerebrospinal fluid barrier.

Mol Immunol 2013 Jan 13;53(1-2):43-51. Epub 2012 Jul 13.

Department of Neurology, The Affiliated Sanming First Hospital of Fujian Medical University, Sanming 365000, Fujian, China.

Experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS), is a T cell-mediated autoimmune condition characterized by prominent inflammation in the CNS. In this model, autoreactive T cells are primed in peripheral lymph nodes and migrate into uninflamed CNS across blood-cerebrospinal fluid barrier (BCSFB) and blood-brain barrier (BBB) to initiate inflammation. However, the molecular mechanism controlling T cell migration remains to be determined. In an in vivo EAE mouse model, we have shown that Irgm1 (also known as LRG-47), a member of the immunity-related GTPase family, promotes the disruption of both BCSFB and BBB, and exacerbates the phenotypes of MOG-induced EAE. During EAE, Irgm1 was up-regulated in reactive astrocytes, ependymal cells and epithelial cells of the choroids plexus, which, in turn, promotes T cell infiltration into the CNS. Electron microscopy study showed that the MOG-induced disruption of both BBB and BCSFB was protected in the Irgm1(-/-) mice. Moreover, the expression of Claudin-5 (CLN-5), a major molecular determinant of BBB, in brain microvessel endothelial cells (BMVECs) was decreased in WT EAE mice while increased in Irgm1(-/-) mice. In addition, the expression of CC-chemokine ligand 20 (CCL-20), an important chemokine mediating lymphocyte trafficking across BCSFB, in the epithelial cells of choroids plexus was significantly suppressed in naïve and EAE-induced Irgm1(-/-) mice. These data suggest that Irgm1 is an important molecular regulator for the properties of both BBB and BCSFB, and a proinflammatory factor for EAE.
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http://dx.doi.org/10.1016/j.molimm.2012.06.009DOI Listing
January 2013

The suppression of CRMP2 expression by bone morphogenetic protein (BMP)-SMAD gradient signaling controls multiple stages of neuronal development.

J Biol Chem 2010 Dec 6;285(50):39039-50. Epub 2010 Oct 6.

Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

The formation of the functional mammalian cerebral cortex requires a concerted control of neurogenesis, neuronal migration, and neuronal morphogenesis. However, molecular mechanisms that control these processes are not well understood. We have found that the BMP signaling downstream transcription factor SMAD1 and CRMP2 (collapsin response mediator protein-2) are inversely and complementarily expressed in the developing neocortex. BMPs can suppress CRMP2 expression in cortical cells. Our ChIP assay demonstrates that both SMAD1 and -4 bind to CRMP2 promoter in the neocortex, and overexpression of SMAD1 and 4 in vivo suppresses CRMP2 expression. RNA interference of CRMP2 and overexpression of dominant negative forms of CRMP2 in utero cause accumulation of multipolar cells in the ventricular, subventricular, and intermediate zones and suppresses neurite outgrowth, suggesting that CRMP2 is required for multipolar to bipolar transition for directional neuronal migration and neurite outgrowth. Thus, our study reveals a novel mechanism that the BMP-SMAD signaling pathway controls neuronal migration and neurite outgrowth by suppressing the transcription of CRMP2.
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http://dx.doi.org/10.1074/jbc.M110.168351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2998125PMC
December 2010

Targeted deletion of numb and numblike in sensory neurons reveals their essential functions in axon arborization.

Genes Dev 2005 Jan 14;19(1):138-51. Epub 2004 Dec 14.

Department of Pathology, University of California San Francisco, San Francisco, California 94143, USA.

Mouse Numb homologs antagonize Notch1 signaling pathways through largely unknown mechanisms. Here we demonstrate that conditional mouse mutants with deletion of numb and numblike in developing sensory ganglia show a severe reduction in axonal arborization in afferent fibers, but no deficit in neurogenesis. Consistent with these results, expression of Cre recombinase in sensory neurons from numb conditional mutants results in reduced endocytosis, a significant increase in nuclear Notch1, and severe reductions in axon branch points and total axon length. Conversely, overexpression of Numb, but not mutant Numb lacking alpha-adaptin-interacting domain, leads to accumulation of Notch1 in markedly enlarged endocytic-lysosomal vesicles, reduced nuclear Notch1, and dramatic increases in axonal length and branch points. Taken together, our data provide evidence for previously unidentified functions of Numb and Numblike in sensory axon arborization by regulating Notch1 via the endocytic-lysosomal pathways.
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http://dx.doi.org/10.1101/gad.1246005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC540232PMC
January 2005