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Ependymal Cells Require Anks1a for Their Proper Development

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Ependymal Cells Require Anks1a for Their Proper Development Molecules and Cells Minireview Ependymal Cells Require Anks1a for Their Proper Development Sunjung Park, Haeryung Lee, Jiyeon Lee, Eunjeong Park, and Soochul Park* Department of Biological Sciences, Sookmyung Women’s University, Seoul 04310, Korea *Correspondence: [email protected] http://dx.doi.org/10.14348/molcells.2018.0432 www.molcells.org Ependymal cells constitute the multi-ciliated epithelium, during embryonic and perinatal brain development (Krieg- which lines the brain ventricular lumen. Although ependymal stein and Alvarez-Buylla, 2009). During postnatal brain de- cells originate from radial glial cells in the perinatal rodent velopment, RGCs gradually transform into terminally differ- brain, the exact mechanisms underlying the full differentia- entiated cell types, such as ependymal cells and astrocytes, tion of ependymal cells are poorly understood. In this report, although some remain as RGC-like neural stem cells in spa- we present evidence that the Anks1a phosphotyrosine bind- tially restricted brain regions (Ihrie and Alvarez-Buylla, 2011). ing domain (PTB) adaptor is required for the proper develop- This developmental differentiation of RGCs into various cell ment of ependymal cells in the rodent postnatal brain. types in the ventricular zone (VZ) and subventricular zone Anks1a gene trap targeted LacZ reporter analysis revealed (SVZ) is a dynamic, finely tuned process. This process may that Anks1a is expressed prominently in the ventricular region change in response to various physiological and environmen- of the early postnatal brain and that its expression is restricted tal stimuli (Manzini and Walsh, 2011). Abnormalities in the- to mature ependymal cells during postnatal brain develop- se developmental dynamics of RGCs during postnatal brain ment. In addition, Anks1a-deficient ependymal cells were development may eventually lead to imbalance in the pro- shown to possess type B cell characteristics, suggesting that duction and connectivity of neurons, which is an underlying ependymal cells require Anks1a in order to be fully differenti- cause of developmental brain disorders such as autism and ated. Finally, Anks1a overexpression in the lateral wall of the schizophrenia (Neale et al., 2012; Ohata and Alvarez-Buylla, neonatal brain resulted in an increase in the number of epen- 2016). dymal cells during postnatal brain development. Altogether, The adult brain ventricles are lined with a layer of ependy- our results suggest that ependymal cells require Anks1a PTB mal cells, which are multi-ciliated and play an essential role adaptor for their proper development. in facilitating the flow of cerebrospinal fluid (CSF) through the ventricular system (Ohata and Alvarez-Buylla, 2016). Keywords: Anks1a, brain development, ependymal cells, PTB Although ependymal cells are generated mainly from RGCs adaptor during embryonic brain development, their maturation be- gins with caudorostral and ventrodorsal direction along the lateral wall (LW) of the lateral ventricle (LV) during the first INTRODUCTION postnatal week (Spassky et al., 2005). Expression of the forkhead transcription factor FoxJ1 is required for the differ- Radial glial cells (RGCs) play pivotal roles in generating new entiation of RGCs into ependymal cells in the postnatal neurons and providing a scaffold for neuronal migration mouse brain (Jacquet et al., 2009). Target genes regulated Received 22 November, 2018; revised 15 January, 2019; accepted 31 January, 2019; published online 12 February, 2019 eISSN: 0219-1032 The Korean Society for Molecular and Cellular Biology. All rights reserved. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/. Mol. Cells 2019; 42(3): 245-251 245 Role of Anks1a in Ependymal Development Sunjung Park et al. by FoxJ1 encode microtubule-associated proteins implicated All mice were handled in accordance with the institutional in ciliogenesis and cell junctional proteins. In addition, the guidelines approved by the Sookmyung Women’s University homeobox transcription factor Six3 is necessary for ependy- Animal Care and Use Committee. mal cell maturation in the postnatal brain through suppress- ing RGC characteristics (Lavado and Oliver, 2011). Evidence X-gal staining and immunohistochemistry indicates that ependymal cells inherit the planar cell polarity For X-gal staining, embryos or brains were dissected in (PCP) set-up of the primary cilia in RGCs (Mirzadeh and Han phosphate-buffered saline (PBS), fixed in 0.2% glutaralde- et al., 2010). Subsequently, both intracellular PCP signaling hyde for 10 min, washed three times with PBS, and subject- and hydrodynamic forces exerted by the emerging CSF flow ed to staining as previously described (Noh et al., 2016). For are critical for the docking and rotational orientation of mo- immunohistochemical analysis, brains were collected from tile cilia in mature ependymal cells (Guirao et al., 2010). mice at the indicated postnatal stages, fixed in 4% para- Therefore, developmental defects in ependymal cells cause formaldehyde, washed in PBS, immersed sequentially in hydrocephalus because the coordinated beating of the mul- 30% sucrose and 30% sucrose/50% OCT at 4℃, and finally ti-cilia and the subsequent stereotypical flow of the CSF are embedded in 100% OCT at -20℃. The embedded brains disturbed (Tissir et al., 2010). After complete differentiation, were cut into 25-μm sections and collected on coated slides. multi-ciliated ependymal cells do not divide any more under The sections were washed, blocked at room temperature, normal conditions. However, they may function as adult and incubated with primary antibodies overnight at 4℃. neural stem cells only in response to brain injury such as Sections were further incubated with species-specific sec- stroke (Carlen et al., 2009). ondary antibodies for 2 h and then washed prior to mount- Anks1a is a cytosolic adaptor with ankyrin repeats, two ing with Hoechst in VECTASHIELD (Vector Laboratories). sterile alpha motifs (SAM), and a phosphotyrosine binding domain (PTB) (Shin et al., 2007; Uhlik et al., 2005). The role Dissection and staining of the LW of Anks1a has been mainly investigated in the signaling For dissection of the LW, mice at the indicated postnatal pathway downstream of the epidermal growth factor recep- stages were perfused with PBS containing 4% paraformal- tor and Eph receptor (Kim et al., 2010; Shin et al., 2007; dehyde and the brains were removed from the skull and Tong et al., 2013). Importantly, a recent report indicated fixed overnight at 4℃. Brains were further manipulated to that Anks1a plays a role in facilitating the export of Eph re- reveal the lateral wall as previously described (Mirzadeh and ceptor tyrosine kinases (RTKs) from the endoplasmic reticu- Doetschet et al., 2010) and the whole mount LWs were lum (ER)(Lee et al., 2016). However, the role of Anks1a in washed three times in PBS containing 0.3% Triton X-100 for vivo, specifically, in the development of ependymal cells and 10 min each. the SVZ niche, has been barely studied to date. Notably, For staining of the LW, the whole mount tissue was Numb and Ank3 have been implicated in the proper devel- blocked in PBS containing 10% horse serum and 0.3% Tri- opment of ependymal cells and the SVZ niche during post- ton X-100 for 30 min and incubated with the primary anti- natal brain development (Kuo et al., 2006; Paez-Gonzalez et bodies overnight. The whole mount tissue was further incu- al., 2011). Numb contains a PTB domain but not ankyrin bated with species-specific secondary antibodies and repeats and SAM domain, whereas Ank3 contains ankyrin Hoechst, washed in PBS, and mounted in VECTASHIELD repeats but not SAM and PTB domains. It was also shown (Vector Laboratories). that expression of the PTB domain-deleted Anks1a inter- rupted the proper development of the ependymal tissue and DNA electroporation into neonatal brain subsequently induced a severe hydrocephalic phenotype The indicated IRES-GFP expression constructs were purified (Park et al., 2015). In this aspect, it is interesting to speculate using EndoFree Plasmid Kit (Quiagen Maxiprep Kit, cat.no. that both ankyrin repeats and the PTB domain may play dis- 12362) according to the manufacturer’s instruction. For tinct roles in the development of the VZ-SVZ during postna- electroporation, DNA solutions (final concentration 2 μg/μl) tal brain development. containing 1% Fast Green were prepared in glass capillaries. In this study, we used Anks1a-deficient mice to demon- The glass capillary was inserted into the lumen of the lateral strate that Anks1a plays a pivotal role in the proper devel- ventricle and 1 μl of DNA solution was slowly injected as opment of ependymal cells during postnatal brain develop- described (Feliciano et al., 2013). The injected pups were ment. placed on the 37℃ heating plate for 15 min and returned to their mother. After 15 days, brains were extracted and sub- MATERIALS AND METHODS jected to immunohistochemical staining. Anks1a mutant mice and bacterial artificial chromosome Antibodies (BAC) transgenic mice The polyclonal chicken GFP antibody used in this study was Anks1a gene-trap mutant mice have been described previ- purchased from Abcam, while the polyclonal rabbit Anks1a ously (Kim et al., 2010). For green fluorescent protein (GFP) antibody used was purchased from Bethyl Laboratories. expression analysis,
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