Knockdown Of JIP3 Impairs Neuronal Radial Migration In The Cerebral Cortex

BackgroundThe cerebral cortex is composed of numerous neurons to formed the largest structure of the brain. It is the complex region of the brain with highly organized, six-layer structure. Six distinct layers with different cellular compositions and functions are responsible for the formation of cortical circuits, cognitive function, sensory perception and consciousness. As such, precisely coordinated migration of neurons to their final target areas in the developing embryo, is essential to achieve appropriate lamination and subsequent circuit formation. It has been investigated that cortical neurons originating from the ventricular zone (VZ) and subventricular zone (SVZ) migrate radially along the process fibers of radial glia through the intermedia zone (IZ) to the developing cortical plate (CP). Although a cohort of signaling pathways has been implicated in the regulation of cortical projection neuron migration, the precise molecular mechanisms are just being revealed.JNK-interacting protein 3 (JIP3) is a JNK scaffold that is highly expressed in adult brain. Knockout of JIP3 in mice resulted in the absence of the telencephalic commissure. Furthermore, it has been reported that JIP3 regulated cancer cell motility through focal adhesion kinase (FAK). However, the role of JIP3 in neuronal migration in developing cortex remains unknown. In this study, we proved the function of JIP3 in neuronal migration by knocking down expression of JIP3 with in utero electroporation. Loss of JIP3 impaired neuronal migration and the movement of neurons was delayed. Furthermore, we investigated the potential mechanism underlying this event and found that KBD and LZ domains were the necessary domains for JIP3 regulating neuronal migration. Taken together, our data suggested that JIP3 acts as a crucial regulator of cortical neuronal migration in the developing cortex.Objective1. To explore the relationship between JIP3 and cerebral cortex development, and identify the role of JIP3 in neuronal migration.2. To investigate the potential mechanism underlying JIP3 regulating neuronal migration, and provide a new target to understanding cerebral cortex development.Methods1. Western BlotCerebral cortex of mice brain from different developmental stage was dissected and homogenized. Samples were boiled at 95℃ for 5min, then subjected to SDS-PAGE for 1h and transferred to a polyvinylidene difluoride membrane. Membranes were blocked by non-fat milk and incubated with primary antibodies and second antibodies. Finally, membranes were detected using the ECL chemiluminescence system. Immunareactive bands were scanned and density quantification was analyzed using NIH ImagJ software.2. In Utero Electroporation.A timed-pregnant mouse was anesthetized with an intraperioneal injection of Pelltobarbitalum Natricum. Five to ten minutes after injection, put the mouse on a working plate with abdomen upside. Their uterine horns were exposed with a midline laparotomy incision. Embryos were taken out and carefully placed on humidified gauze pads. The uterus was squeezed gently to push embryos closer to the uterine wall. Plasmid DNA plus 0.01% Fast Green was injected into the lateral ventricles of the embryonic brain with the glass micropipette. A volume of 2μLof shRNA plasmids (1.5 μg/μL) or scramble constructs (1.5μg/μL) were injected. For rescue experiments, expression constructs were coinjected with shRNA or scramble plasmids. For electroporation,5 × 50 ms,30 V square pulses separated by 950 ms intervals were delivered with forceps-type electrodes connected to an electroporator. The uterus was then replaced into the abdominal cavity, and the abdomen wall and skin were sutured using the surgical needle and thread. The whole procedure was completed within 40 min. The pregnant mouse was warmed in an incubator until it became conscious, and embryos were allowed developed in utero for the time indicated.3. Plasmid constructsSmall interference RNA sequences targeting JIP3 were inserted downstream of the U6 promoter, and the target sequence was as follows: rat and mouse JIP3, CAG GCC GAG GAG AAA TTC A. The efficiency of knockdown was assessed by transfecting with the resulting siRNA or the scramble siRNA in HEK-293 cells and analyzed by Western blot. The EGFP-tagged JIP3-resistant plasmid was constructed using site-directed mutagenesis without altering the amino acid sequence, and the JIP3 target sequences were mutated to the following: CAa GCg GAa GAG AAg TTC A. pCAGIG-IRES-EGFP was chosen as the vector for the constructs used forin utero electroporation with the cytomegalovirus (CMV) early enhancer element and chicken beta-actin promoter-enhanced green fluorescent protein (EGFP).4. ImmunostainingBrains were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight, then placed in 30% sucrose in phosphate buffer saline (pH 7.4) overnight, and frozen for sectioning with a Leica cryostat. Immunostaining was performed with standard protocols involving overnight incubation with primary antibodies at 4℃ and incubation with appropriate fluorescent secondary antibodies for 1 h at room temperature.Results:1. JIP3 participated in cerebral cortex development1.1 The expression profile of JIP3 in developing cortex JIP3 is highly expressed in embryonic and postnatal cortex. Especially in E13 and P0, protein level has obvious increased.1.2 Knockdown JIP3 impaired cortical six-layer structure Conditional knockdown JIP3 in telencephalon from E11 caused the thickness of cortex thinner and the six-layer structure of cortex turned disordered compared to wide-type mouse in adult mouse brain. In embryonic, the six-layer structure of cortex was also disordered.2. Loss of JIP3 impaired neuronal migration2.1 Loss of JIP3 had no influence on neuronal polarization We analysed morphology of neurons in IZ, accouted the percentage of bipolar or multipolar neurons, and found that siJIP3 has no effect on neuronal morphology.2.2 Knockdown JIP3 had no influence on proliferation We detected the effects of JIP3 on proliferation by labeling newborn cells with BrdU, and no significant differences were found between scramble group and siRNA group.2.3 Knockdown JIP3 impaired neuronal migration siRNA knocked down JIP3 expression efficiently. Additionally, the movement of neurons was obviously disrupted by loss of JIP3. The migrating neurons were mainly stayed in intermediate zone after knockdown of JIP3.3. Loss of JIP3 delayed neuronal migration After in utero eletroporation at E15.5, we dissected the brain at PO and P7. At P0, the percentage of neurons in layer II-IV was largely less in siJIP3 group and neurons were mainly stayed in layer VI and VZ/SVZ. But after 7 days migration, 70% of neurons reached the up layers.4. The mechanism of JIP3 regulating neuronal migration We constructed three JIP3 mutant plasmids, Res-JIP3AJBD, Res-JIP3△KBDALZ and Res-JIP3△CC1. We co-injected different JIP3 mutant plasmids with siJIP3 plasmid, and detected changes in the impaired migration caused by siJIP3. We found that KBD, LZ and CC1 were necessary for JIP3 regulating neuronal migration.Conlusion:1. Our study revealed the precious expression profile of JIP3 in developing cortex. Loss of function of JIP3 from embryonic, the structure of cerebral cortex was disordered.2. Knockdown of JIP3 delayed neuronal migration without disruped neuronal morphology and proliferation.3. KBD and LZ domains were important for JIP3 regulating neuronal migration.Innovation:1. we deciphered the precious expression of JIP3 both in embryonic and postnatal periods;2. We found knockdown JIP3 delayed neuronal migration;3. KBD and LZ domains were important for JIP3 regulating neuronal migration.