NEDL2 is an essential regulator of enteric neural development and GDNF/Ret signaling Rongfei Wei, Xiao Qiu, Shaoxia Wang, Yang Li, Yiwu Wang, Kefeng Lu, Yesheng Fu, Guichun Xing, Fuchu He, Lingqiang Zhang PII: DOI: Reference:
S0898-6568(14)00415-X doi: 10.1016/j.cellsig.2014.12.013 CLS 8362
To appear in:
Cellular Signalling
Received date: Revised date: Accepted date:
25 October 2014 3 December 2014 23 December 2014
Please cite this article as: Rongfei Wei, Xiao Qiu, Shaoxia Wang, Yang Li, Yiwu Wang, Kefeng Lu, Yesheng Fu, Guichun Xing, Fuchu He, Lingqiang Zhang, NEDL2 is an essential regulator of enteric neural development and GDNF/Ret signaling, Cellular Signalling (2014), doi: 10.1016/j.cellsig.2014.12.013
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT NEDL2 is an essential regulator of enteric neural development and GDNF/Ret signaling
IP
T
Rongfei Wei1,2, Xiao Qiu1,2, Shaoxia Wang4, Yang Li4, Yiwu Wang2, Kefeng
SC R
Lu2,Yesheng Fu2, Guichun Xing2, Fuchu He1,2,*, Lingqiang Zhang2,3,*
School of Life Sciences, Tsinghua University, Beijing 100084, China.
2
State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing
NU
1
MA
Institute of Radiation Medicine, Collaborative Innovation Center for Cancer Medicine, Beijing 100850, China.
116044, China.
Department of Experimental Pathology, Beijing Institute of Radiation Medicine,
CE P
4
D
Institute of Cancer Stem Cell, Dalian Medical University, Dalian, Liaoning Province,
TE
3
Beijing 100850, China
AC
*Correspondence should be addressed to L.Z. (email: [email protected]) or F.H. (e-mail: [email protected])
1
ACCEPTED MANUSCRIPT Although glial cell line-derived neurotrophic factor (GDNF)/Ret signaling is essential for enteric nervous system (ENS) development, the positive regulators
IP
T
regulating GDNF/Ret signaling and controlling ENS development are poorly
SC R
understood. Here, we show that NEDD4-related E3 ubiquitin ligase-2 (NEDL2), plays an essential and positive physiological role in regulating ENS development and GDNF/Ret signaling. All of the NEDL2-deficient mice die within 2 weeks
NU
after birth, showing low body weight. These mice showed a progressive bowel
MA
motility defect resulting from intestinal aganglionosis. We show that NEDL2 positively regulates enteric neural precursor proliferation through the
TE
function of NEDL2 in vivo.
D
GDNF/Akt signaling pathway. Together, these findings unveil the physiological
CE P
Keywords: Enteric nervous system; NEDL2; GDNF/Ret signaling; Mouse
AC
1. Introduction
The enteric nervous system (ENS) is a complex network of neurons and glia within the wall of the intestine, and it regulates intestinal motility, secretion and blood flow [1,2]. Cells forming the ENS derive from the neural crest and migrate along the intestine [3]. In mice, around embryonic day (E) 9-9.5, vagal crest-derived cells enter the foregut and then migrate caudally, and by E15 the enteric neural crest-derived cells (ENCCs) colonize the entire gastrointestinal tract [2]. Precise spatiotemporal regulation of ENCCs proliferation, differentiation and migration is essential for ENS development. Studies have elucidated that many transcription factors, secreted ligands
2
ACCEPTED MANUSCRIPT and cell-surface molecules are involved in the regulation of ENS development [4], GDNF, through its receptor Ret, activates the most important pathway in the
IP
T
regulation of ENS development. It has been demonstrated that GDNF/Ret signaling
SC R
regulates multiple aspects of ENS development, including the survival, proliferation, migration, and differentiation of enteric neurons [2]. GDNF- and Ret- deficient mice exhibit absence of enteric neurons as well as kidney agenesis [5,6]. Several regulators
NU
of the GDNF/Ret pathway have been identified, including Sprouty1, Sprouty2 and
MA
KIF26A. Mice lacking Sprouty1 exhibit kidney and urinary tract anomalies and mice lacking Sprouty2 or KIF26A show hyperganlionosis [7-9].
D
NEDL2 (also known as HECW2) belongs to the Nedd4 family of E3 ubiquitin
TE
ligases [10]. Members of the Nedd4 family play important roles in both physiological
CE P
and pathological processes. For example, Smurf1 suppresses the osteogenic activity of osteoblasts by inhibiting BMP signaling [11]. Nedd4-2 is critical for the regulation of
AC
sodium ion channels [12]. WWP2 controls craniofacial development [13], and Itch regulates T cell differentiation [14]. In addition, Smurf2 and WWP2 have been shown to play important roles in tumorigenesis [15,16]. However, the physiological functions of NEDL2 remain poorly understood. Here we show that global ablation of NEDL2 in C57/B6J mice leads to defective development of the ENS. This study provides the first evidence for the physiological function of NEDL2 in vivo.
2. Material and Methods 2.1. Gene targeting of NEDL2 A targeting vector was constructed to disrupt the NEDL2 gene. Mice carrying the
3
ACCEPTED MANUSCRIPT targeted NEDL2 allele were bred with EIIa-Cre transgenic mice to remove the neo gene and generate NEDL2LacZ/+ reporter mice in which exons 5-7 are deleted and
IP
T
replaced by an IRES-LacZ gene. Offspring were intercrossed to generate homozygote
SC R
NEDL2LacZ/LacZ (NEDL2-/-) mice.
2.2. X-gal histochemistry
NU
Tissue frozen sections were fixed in fresh 4% paraformaldehyde for 15 min at ice.
MA
Sections were rinsed with wash buffer (100 mM sodium phosphate (pH 7.3), 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40) three times, for thirty minutes
D
each, at room temperature. Sections were then stained in the final X-gal solution at
TE
37 °C until color appeared. They were then washed three times in PBS,
CE P
counter-stained with Nuclear Fast Red and mounted in resinene.
AC
2.3. Cell proliferation assay
Three hours before sacrifice, mice were injected intraperitoneally with BrdU (Sigma) at 100 mg/kg of body weight. Intestines dissected from the embryos at E12.5 were fixed in 10% formalin solution for approximately 24 hours, embedded in paraffin wax and sectioned longitudinally at 3μm. Then, sections were incubated with anti-BrdU antibody (Cell Signaling Technology) overnight at 4°C. Signal amplification and detection was performed using TRITC-conjugated secondary antibodies according to the manufacturer’s instructions. Sections were counterstained with DAPI (Sigma) to identify nuclei. Confocal microscopy was performed on a Zeiss
4
ACCEPTED MANUSCRIPT LSM 510 Meta. Four newborn animals for each genotype were analyzed. The positive cells were counted in at least three randomly selected images from every section. We
IP
T
then compared numbers of BrdU-positive cells using the unpaired t test (P
S0898-6568(14)00415-X doi: 10.1016/j.cellsig.2014.12.013 CLS 8362
To appear in:
Cellular Signalling
Received date: Revised date: Accepted date:
25 October 2014 3 December 2014 23 December 2014
Please cite this article as: Rongfei Wei, Xiao Qiu, Shaoxia Wang, Yang Li, Yiwu Wang, Kefeng Lu, Yesheng Fu, Guichun Xing, Fuchu He, Lingqiang Zhang, NEDL2 is an essential regulator of enteric neural development and GDNF/Ret signaling, Cellular Signalling (2014), doi: 10.1016/j.cellsig.2014.12.013
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT NEDL2 is an essential regulator of enteric neural development and GDNF/Ret signaling
IP
T
Rongfei Wei1,2, Xiao Qiu1,2, Shaoxia Wang4, Yang Li4, Yiwu Wang2, Kefeng
SC R
Lu2,Yesheng Fu2, Guichun Xing2, Fuchu He1,2,*, Lingqiang Zhang2,3,*
School of Life Sciences, Tsinghua University, Beijing 100084, China.
2
State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing
NU
1
MA
Institute of Radiation Medicine, Collaborative Innovation Center for Cancer Medicine, Beijing 100850, China.
116044, China.
Department of Experimental Pathology, Beijing Institute of Radiation Medicine,
CE P
4
D
Institute of Cancer Stem Cell, Dalian Medical University, Dalian, Liaoning Province,
TE
3
Beijing 100850, China
AC
*Correspondence should be addressed to L.Z. (email: [email protected]) or F.H. (e-mail: [email protected])
1
ACCEPTED MANUSCRIPT Although glial cell line-derived neurotrophic factor (GDNF)/Ret signaling is essential for enteric nervous system (ENS) development, the positive regulators
IP
T
regulating GDNF/Ret signaling and controlling ENS development are poorly
SC R
understood. Here, we show that NEDD4-related E3 ubiquitin ligase-2 (NEDL2), plays an essential and positive physiological role in regulating ENS development and GDNF/Ret signaling. All of the NEDL2-deficient mice die within 2 weeks
NU
after birth, showing low body weight. These mice showed a progressive bowel
MA
motility defect resulting from intestinal aganglionosis. We show that NEDL2 positively regulates enteric neural precursor proliferation through the
TE
function of NEDL2 in vivo.
D
GDNF/Akt signaling pathway. Together, these findings unveil the physiological
CE P
Keywords: Enteric nervous system; NEDL2; GDNF/Ret signaling; Mouse
AC
1. Introduction
The enteric nervous system (ENS) is a complex network of neurons and glia within the wall of the intestine, and it regulates intestinal motility, secretion and blood flow [1,2]. Cells forming the ENS derive from the neural crest and migrate along the intestine [3]. In mice, around embryonic day (E) 9-9.5, vagal crest-derived cells enter the foregut and then migrate caudally, and by E15 the enteric neural crest-derived cells (ENCCs) colonize the entire gastrointestinal tract [2]. Precise spatiotemporal regulation of ENCCs proliferation, differentiation and migration is essential for ENS development. Studies have elucidated that many transcription factors, secreted ligands
2
ACCEPTED MANUSCRIPT and cell-surface molecules are involved in the regulation of ENS development [4], GDNF, through its receptor Ret, activates the most important pathway in the
IP
T
regulation of ENS development. It has been demonstrated that GDNF/Ret signaling
SC R
regulates multiple aspects of ENS development, including the survival, proliferation, migration, and differentiation of enteric neurons [2]. GDNF- and Ret- deficient mice exhibit absence of enteric neurons as well as kidney agenesis [5,6]. Several regulators
NU
of the GDNF/Ret pathway have been identified, including Sprouty1, Sprouty2 and
MA
KIF26A. Mice lacking Sprouty1 exhibit kidney and urinary tract anomalies and mice lacking Sprouty2 or KIF26A show hyperganlionosis [7-9].
D
NEDL2 (also known as HECW2) belongs to the Nedd4 family of E3 ubiquitin
TE
ligases [10]. Members of the Nedd4 family play important roles in both physiological
CE P
and pathological processes. For example, Smurf1 suppresses the osteogenic activity of osteoblasts by inhibiting BMP signaling [11]. Nedd4-2 is critical for the regulation of
AC
sodium ion channels [12]. WWP2 controls craniofacial development [13], and Itch regulates T cell differentiation [14]. In addition, Smurf2 and WWP2 have been shown to play important roles in tumorigenesis [15,16]. However, the physiological functions of NEDL2 remain poorly understood. Here we show that global ablation of NEDL2 in C57/B6J mice leads to defective development of the ENS. This study provides the first evidence for the physiological function of NEDL2 in vivo.
2. Material and Methods 2.1. Gene targeting of NEDL2 A targeting vector was constructed to disrupt the NEDL2 gene. Mice carrying the
3
ACCEPTED MANUSCRIPT targeted NEDL2 allele were bred with EIIa-Cre transgenic mice to remove the neo gene and generate NEDL2LacZ/+ reporter mice in which exons 5-7 are deleted and
IP
T
replaced by an IRES-LacZ gene. Offspring were intercrossed to generate homozygote
SC R
NEDL2LacZ/LacZ (NEDL2-/-) mice.
2.2. X-gal histochemistry
NU
Tissue frozen sections were fixed in fresh 4% paraformaldehyde for 15 min at ice.
MA
Sections were rinsed with wash buffer (100 mM sodium phosphate (pH 7.3), 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40) three times, for thirty minutes
D
each, at room temperature. Sections were then stained in the final X-gal solution at
TE
37 °C until color appeared. They were then washed three times in PBS,
CE P
counter-stained with Nuclear Fast Red and mounted in resinene.
AC
2.3. Cell proliferation assay
Three hours before sacrifice, mice were injected intraperitoneally with BrdU (Sigma) at 100 mg/kg of body weight. Intestines dissected from the embryos at E12.5 were fixed in 10% formalin solution for approximately 24 hours, embedded in paraffin wax and sectioned longitudinally at 3μm. Then, sections were incubated with anti-BrdU antibody (Cell Signaling Technology) overnight at 4°C. Signal amplification and detection was performed using TRITC-conjugated secondary antibodies according to the manufacturer’s instructions. Sections were counterstained with DAPI (Sigma) to identify nuclei. Confocal microscopy was performed on a Zeiss
4
ACCEPTED MANUSCRIPT LSM 510 Meta. Four newborn animals for each genotype were analyzed. The positive cells were counted in at least three randomly selected images from every section. We
IP
T
then compared numbers of BrdU-positive cells using the unpaired t test (P
