Defining the Role of the Mouse Jhy Gene in the Ependyma and Choroid Plexus

The cerebrospinal fluid (CSF) is produced by the choroid plexus and is contained within the brain ventricular system (Damkier et al., 2013). CSF flows from the lateral ventricles to the third ventricle, where it then enters the fourth ventricle through the cerebral aqueduct (Brinker et al., 2014). The composition of the CSF is derived from passive filtration of plasma and membrane secretion and it has three mains functions: 1) protect the brain by serving as a cushion against mechanical shock, 2) serve as a route for nutrient delivery and signaling factors, and 3) carry waste products and toxins away from the brain (Lun et al., 2015; Spector et al., 2015). CSF production and circulation should be carefully regulated to allow proper brain development (Gato et al., 2014; Mohammad Nabiuni, 2015). Increased CSF volume causes ventricular dilation and it can lead to the development of a neurological condition known as hydrocephalus. Congenital hydrocephalus is the most common form of the disease, which is present at birth and it affects 1-2/1000 children in the United States (Kahle et al., 2015). Hydrocephalus can result from abnormalities in the production, flow or absorption of the CSF, and if untreated it can lead to death. The ventricles of the brain are lined by a monolayer of ciliated squamous epithelia known as the ependymal cells. The ependyma carry motile cilia (9+2), which coordinately beat to produce a laminar flow and promote CSF circulation throughout the ventricles (Spassky, 2013). The choroid plexus is composed of a monolayer of modified ependymal cells though these cells have developed a unique polarization of membrane associated proteins, along with other secretory properties that allow choroid plexus (CP) cells to function in CSF secretion (Damkier et al., 2013; Dziegielewska et al., 2001). Hydrocephalus can result from the disruption of ependymal cells and/or the choroid plexus by the loss of CSF flow and CSF overproduction, respectively (Baas et al., 2006a; Banizs et al., 2005). Despite the undeniable importance of the ependyma and the CP in CSF homeostasis, the mechanisms and factors involved in their differentiation are largely unknown. The work presented here aimed to further characterize the structure and function of the ependymal cells and the specialized ependymal cells of the CP. The JhylacZ mouse line carries an insertional mutation in the Jhy gene (formerly 4931429I11Rik), and homozygous JhylacZ/lacZ mice develop a rapidly progressive juvenile hydrocephalus (Appelbe et al., 2013). Molecular analysis of the ependymal cells in JhylacZ/lacZ mice was performed using a cell-type specific marker approach to assess the expression of markers involved in vital cellular processes of these cells. JhylacZ/lacZ mice display abnormal ependymal cell differentiation with ventricular ependyma retaining an unorganized and multi-layered morphology, representative of immature ependymal cells. Morphological and molecular analysis of the ependyma demonstrated a delay rather than a block in differentiation. Additionally, JhylacZ/lacZ ependymal cells manifest disruptions in adherens junction formation. Ultrastructural analysis of postnatal JhylacZ/lacZ mice found abnormal organization of the motile cilia lining the lateral ventricles of the brain, structures believed to be required for proper CSF flow. JhylacZ/lacZ ependymal cells have defects in the polarized organization of the apically located cilia. The latter resulted in severely reduced motility, a likely cause for the development of hydrocephalus. A second Jhy mutant model (i.e. JhylacZNeo ) was generated upon the availability of Jhy targeted embryonic stem cells from the Knockout mouse consortium (KOMP). We sought to identify the role of Jhy in the specialized ependymal cells of the choroid plexus in the JhylacZNeo mouse line. Our analysis indicates that JhylacZNeo/lacZNeo develop early onset hydrocephalus, with mice rarely surviving past 3 weeks of age. JhylacZNeo/lacZNeo CP also displays disruptions in adherens junction formation, with abnormal localization of the key adherens junction protein, E-cadherin. TEM analysis of the CP in JhylacZNeo/lacZNeo demonstrated defects in ciliary ultrastructure, along with altered microvilli distribution. Together, this data identifies Jhy as a gene required for proper adherens junction formation and cilia ultrastructure, in the ependyma and the specialized ependyma of the choroid plexus.