Choroid Plexus Development Research Articles

The normal 14-18 gestational weeks "parasagittal complex" view of the fetal brain. A 3D transvaginal neurosonographic study.

To study the early second trimester development of brain hemispheres, lateral ventricles, choroid plexus, and ganglionic eminence/basal ganglia complex (GEBG). A retrospective analysis of TVUS 3D volumes of 14-18 gestational weeks (GW) fetuses. Hemispheres were analyzed for wall thickness, choroid plexus extension, GEBG height and length, lamination pattern (intermediate zone and the subplate border, IZ-SP), ventricle height, width, and angle. Measurements were correlated with GW and assessed for symmetry and impact of probe resolution. We included 84 fetuses (168 hemispheres). The CP location is variable at 14-16GW, becoming consistently and symmetrically posterior at 18GW. Hemispheric thickness, GEBG height and length grow significantly with fetal age, whereas ventricle height, width, and angle regress. The detection rate of the IZ-SP line at 14, 15, 16, 17, and 18weeks was 0%, 24%, 78.26%, 100%, and 100%, respectively. The ratio between the upper and lower segments of the cerebral lamination grows with GW. For all brain structures, the asymmetry between sides was significant only for ventricular height. The transducer type did not have a significant effect on any outcome except for ventricle height. These normal features of the parasagittal view should aid clinicians in fetal brain assessment during the early weeks of the second trimester.

Choroid plexus mis-splicing and altered cerebrospinal fluid composition in myotonic dystrophy type 1.

Myotonic dystrophy type 1 is a dominantly inherited multisystemic disease caused by CTG tandem repeat expansions in the DMPK 3' untranslated region. These expanded repeats are transcribed and produce toxic CUG RNAs that sequester and inhibit activities of the MBNL family of developmental RNA processing factors. Although myotonic dystrophy is classified as a muscular dystrophy, the brain is also severely affected by an unusual cohort of symptoms, including hypersomnia, executive dysfunction, as well as early onsets of tau/MAPT pathology and cerebral atrophy. To address the molecular and cellular events that lead to these pathological outcomes, we recently generated a mouse Dmpk CTG expansion knock-in model and identified choroid plexus epithelial cells as particularly affected by the expression of toxic CUG expansion RNAs. To determine if toxic CUG RNAs perturb choroid plexus functions, alternative splicing analysis was performed on lateral and hindbrain choroid plexi from Dmpk CTG knock-in mice. Choroid plexus transcriptome-wide changes were evaluated in Mbnl2 knockout mice, a developmental-onset model of myotonic dystrophy brain dysfunction. To determine if transcriptome changes also occurred in the human disease, we obtained post-mortem choroid plexus for RNA-seq from neurologically unaffected (two females, three males; ages 50-70 years) and myotonic dystrophy type 1 (one female, three males; ages 50-70 years) donors. To test that choroid plexus transcriptome alterations resulted in altered CSF composition, we obtained CSF via lumbar puncture from patients with myotonic dystrophy type 1 (five females, five males; ages 35-55 years) and non-myotonic dystrophy patients (three females, four males; ages 26-51 years), and western blot and osmolarity analyses were used to test CSF alterations predicted by choroid plexus transcriptome analysis. We determined that CUG RNA induced toxicity was more robust in the lateral choroid plexus of Dmpk CTG knock-in mice due to comparatively higher Dmpk and lower Mbnl RNA levels. Impaired transitions to adult splicing patterns during choroid plexus development were identified in Mbnl2 knockout mice, including mis-splicing previously found in Dmpk CTG knock-in mice. Whole transcriptome analysis of myotonic dystrophy type 1 choroid plexus revealed disease-associated RNA expression and mis-splicing events. Based on these RNA changes, predicted alterations in ion homeostasis, secretory output and CSF composition were confirmed by analysis of myotonic dystrophy type 1 CSF. Our results implicate choroid plexus spliceopathy and concomitant alterations in CSF homeostasis as an unappreciated contributor to myotonic dystrophy type 1 CNS pathogenesis.

Constitutive activation of canonical Wnt signaling disrupts choroid plexus epithelial fate

The choroid plexus secretes cerebrospinal fluid and is critical for the development and function of the brain. In the telencephalon, the choroid plexus epithelium arises from the Wnt- expressing cortical hem. Canonical Wnt signaling pathway molecules such as nuclear β-CATENIN are expressed in the mouse and human embryonic choroid plexus epithelium indicating that this pathway is active. Point mutations in human β-CATENIN are known to result in the constitutive activation of canonical Wnt signaling. In a mouse model that recapitulates this perturbation, we report a loss of choroid plexus epithelial identity and an apparent transformation of this tissue to a neuronal identity. Aspects of this phenomenon are recapitulated in human embryonic stem cell derived organoids. The choroid plexus is also disrupted when β-Catenin is conditionally inactivated. Together, our results indicate that canonical Wnt signaling is required in a precise and regulated manner for normal choroid plexus development in the mammalian brain.

Correction: MEIS-WNT5A axis regulates development of fourth ventricle choroid plexus.

There was an error in Development (2021) 148, dev192054 (doi:10.1242/dev.192054).In the author affiliations, affiliation 4 was incorrect.Corrected:Karol Kaiser1, Ahram Jang2, Petra Kompanikova1, Melody P. Lun2, Jan Prochazka3, Ondrej Machon4, Neil Dani2, Michaela Prochazkova3, Benoit Laurent5,6, Daniel Gyllborg7, Renee van Amerongen8, Ryann M. Fame2, Suhasini Gupta2, Feizhen Wu9, Roger A. Barker10, Ivana Bukova3, Radislav Sedlacek3, Zbynek Kozmik11, Ernest Arenas12, Maria K. Lehtinen2 and Vitezslav Bryja11Department of Experimental Biology, Faculty of Science, Masaryk University, Brno 61137, Czech Republic.2Department of Pathology, Boston Children's Hospital, Boston, MA 02115, USA.3Czech Centre for Phenogenomics and Laboratory of Transgenic Models of Diseases, Institute of Molecular Genetics of the CAS, Prague 142 20, Czech Republic.4Department of Developmental Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic.5Research Center on Aging, CIUSSS de l'Estrie - CHUS, Sherbrooke, QC 75361, Canada.6Department of Biochemistry and Functional Genomics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC 75281, Canada.7Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna SE-106 91, Sweden.8Swammerdam Institute for Life Sciences, Section of Molecular Cytology, Faculty of Science, University of Amsterdam 1098 XH, Netherlands.9Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China.10John van Geest Centre for Brain Repair and WT-MRC Cambridge Stem Cell Centre, University of Cambridge, Cambridge CB2 0PY, UK.11Laboratory of Transcriptional Regulation, Institute of Molecular Genetics of the CAS, Prague 142 20, Czech Republic.12Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm 171 77, Sweden.Original:Karol Kaiser1, Ahram Jang2, Petra Kompanikova1, Melody P. Lun2, Jan Prochazka3, Ondrej Machon4, Neil Dani2, Michaela Prochazkova3, Benoit Laurent5,6, Daniel Gyllborg7, Renee van Amerongen8, Ryann M. Fame2, Suhasini Gupta2, Feizhen Wu9, Roger A. Barker10, Ivana Bukova3, Radislav Sedlacek3, Zbynek Kozmik4, Ernest Arenas11, Maria K. Lehtinen2 and Vitezslav Bryja11Department of Experimental Biology, Faculty of Science, Masaryk University, Brno 61137, Czech Republic.2Department of Pathology, Boston Children's Hospital, Boston, MA 02115, USA.3Czech Centre for Phenogenomics and Laboratory of Transgenic Models of Diseases, Institute of Molecular Genetics of the CAS, Prague 142 20, Czech Republic.4Laboratory of Transcriptional Regulation, Institute of Molecular Genetics of the CAS, Prague 142 20, Czech Republic.5Research Center on Aging, CIUSSS de l'Estrie - CHUS, Sherbrooke, QC 75361, Canada.6Department of Biochemistry and Functional Genomics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC 75281, Canada.7Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna SE-106 91, Sweden.8Swammerdam Institute for Life Sciences, Section of Molecular Cytology, Faculty of Science, University of Amsterdam 1098 XH, Netherlands.9Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China.10John van Geest Centre for Brain Repair and WT-MRC Cambridge Stem Cell Centre, University of Cambridge, Cambridge CB2 0PY, UK.11Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm 171 77, Sweden.Both the online full-text and PDF versions have been updated.The authors apologise to readers for this error and any inconvenience this may have caused.

OTX2 Homeoprotein Functions in Adult Choroid Plexus.

The choroid plexus is an important blood barrier that secretes cerebrospinal fluid, which essential for embryonic brain development and adult brain homeostasis. The OTX2 homeoprotein is a transcription factor that is critical for choroid plexus development and remains highly expressed in adult choroid plexus. Through RNA sequencing analyses of constitutive and conditional knockdown adult mouse models, we reveal putative functional roles for OTX2 in adult choroid plexus function, including cell signaling and adhesion, and show that OTX2 regulates the expression of factors that are secreted into the cerebrospinal fluid, notably transthyretin. We also show that Otx2 expression impacts choroid plexus immune and stress responses, and affects splicing, leading to changes in the mRNA isoforms of proteins that are implicated in the oxidative stress response and DNA repair. Through mass spectrometry analysis of OTX2 protein partners in the choroid plexus, and in known non-cell-autonomous target regions, such as the visual cortex and subventricular zone, we identify putative targets that are involved in cell adhesion, chromatin structure, and RNA processing. Thus, OTX2 retains important roles for regulating choroid plexus function and brain homeostasis throughout life.

MEIS-WNT5A axis regulates development of fourth ventricle choroid plexus.

The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.

Assessment of common housekeeping genes as reference for gene expression studies using RT-qPCR in mouse choroid plexus

Choroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

A sart1 Zebrafish Mutant Results in Developmental Defects in the Central Nervous System.

The spliceosome consists of accessory proteins and small nuclear ribonucleoproteins (snRNPs) that remove introns from RNA. As splicing defects are associated with degenerative conditions, a better understanding of spliceosome formation and function is essential. We provide insight into the role of a spliceosome protein U4/U6.U5 tri-snRNP-associated protein 1, or Squamous cell carcinoma antigen recognized by T-cells (Sart1). Sart1 recruits the U4.U6/U5 tri-snRNP complex to nuclear RNA. The complex then associates with U1 and U2 snRNPs to form the spliceosome. A forward genetic screen identifying defects in choroid plexus development and whole-exome sequencing (WES) identified a point mutation in exon 12 of sart1 in Danio rerio (zebrafish). This mutation caused an up-regulation of sart1. Using RNA-Seq analysis, we identified additional upregulated genes, including those involved in apoptosis. We also observed increased activated caspase 3 in the brain and eye and down-regulation of vision-related genes. Although splicing occurs in numerous cells types, sart1 expression in zebrafish was restricted to the brain. By identifying sart1 expression in the brain and cell death within the central nervous system (CNS), we provide additional insights into the role of sart1 in specific tissues. We also characterized sart1’s involvement in cell death and vision-related pathways.

WNT5a Regulates Epithelial Morphogenesis in the Developing Choroid Plexus.

The choroid plexus (CP) is the predominant supplier of cerebral spinal fluid (CSF) and the site of the blood-CSF barrier and is thus essential for brain development and central nervous system homeostasis. Despite these crucial roles, our understanding of the molecular and cellular processes giving rise to the CPs within the ventricles of the mammalian brain is very rudimentary. Here, we identify WNT5a as an important regulator of CP development, where it acts as a pivotal factor driving CP epithelial morphogenesis in all ventricles. We show that WNT5a is essential for the establishment of a cohesive epithelium in the developing CP. We find that in its absence all CPs are substantially reduced in size and complexity and fail to expand into the ventricles. Severe defects were observed in the epithelial cytoarchitecture of all Wnt5a-/- CPs, exemplified by loss of apicobasally polarized morphology and detachment from the ventricular surface and/or basement membrane. We also present evidence that the WNT5a receptor, RYK, and the RHOA kinase, ROCK, are required for normal CP epithelial morphogenesis. Our study, therefore, reveals important insights into the molecular and cellular mechanisms governing CP development.

Intraventricular Hemorrhage Induces Rapid Calcium Signaling and Transcriptional Changes in the Developing Choroid Plexus

Abstract INTRODUCTION Intraventricular hemorrhage (IVH) leads to acute hydrocephalus in a subset of preterm infants by unclear mechanisms that potentially include increased cerebrospinal fluid (CSF) production by the choroid plexus (ChP), based on recent work in adult rats (Karimy et al., 2017). The earliest physiologic changes in ChP epithelial cells likely involve calcium signaling, which is a known cellular messenger in other secretory epithelial cell populations. The following experiments utilize rapid calcium imaging to evaluate how calcium signaling is regulated in embryonic ChP explants, and additionally determine specific transcriptional alterations in Vivo. METHODS 1) Calcium imaging of embryonic explants: Lateral ventricle ChP is dissected at embryonic day 14.5 (E14.5) from a conditional transgenic mouse line (FoxJ1-Cre; Gcamp6f). The ChP explants are kept viable with artificial CSF, exposed to focal blood injections, and imaged for calcium responses using epifluorescence and two-photon imaging. 2) Transcriptional analysis in a model of preterm IVH: Quantitative RT-PCR is performed on acutely dissected ChP at 24 and 48 hr, following in utero injection of age-matched blood in the lateral ventricles of individual embryos (E14.5 pregnant CD1 mice). RESULTS Upon exposure to age-matched E14.5 plasma, nearly all ChP epithelial cells rapidly release highly concentrated intracellular calcium followed by a mosaic pattern of recurrent calcium release. Using different conditions of calcium blockade, we identify endoplasmic reticulum storage as the primary source of intracellular calcium release. Expression of NKCC1 (the predominant apical ion co-transporter in the ChP) rises by 24 hr, suggesting increased ChP ion permeability, consistent with prior reports of a CSF hypersecretion phenotype. CONCLUSION IVH causes immediate intracellular calcium release by the ChP epithelia, followed by increased NKCC1 transcription within 24 hr. Together, these data suggest a rapid and dynamic embryonic ChP responsiveness to hemorrhage and contributes to our understanding of IVH-induced hydrocephalus pathophysiology.

Development of Circumventricular Organs in the Mirror of Zebrafish Enhancer-Trap Transgenics.

The circumventricular organs (CVOs) are small structures lining the cavities of brain ventricular system. They are associated with the semitransparent regions of the blood-brain barrier (BBB). Hence it is thought that CVOs mediate biochemical signaling and cell exchange between the brain and systemic blood. Their classification is still controversial and development not fully understood largely due to an absence of tissue-specific molecular markers. In a search for molecular determinants of CVOs we studied the green fluorescent protein (GFP) expression pattern in several zebrafish enhancer trap transgenics including Gateways (ET33-E20) that has been instrumental in defining the development of choroid plexus. In Gateways the GFP is expressed in regions of the developing brain outside the choroid plexus, which remain to be characterized. The neuroanatomical and histological analysis suggested that some previously unassigned domains of GFP expression may correspond to at least six other CVOs–the organum vasculosum laminae terminalis (OVLT), subfornical organ (SFO), paraventricular organ (PVO), pineal (epiphysis), area postrema (AP) and median eminence (ME). Two other CVOs, parapineal and subcommissural organ (SCO) were detected in other enhancer-trap transgenics. Hence enhancer-trap transgenic lines could be instrumental for developmental studies of CVOs in zebrafish and understanding of the molecular mechanism of disease such a hydrocephalus in human. Their future analysis may shed light on general and specific molecular mechanisms that regulate development of CVOs.

Effects of alcohol on the development of choroid plexus in chick embryo

Effects of alcohol on the development of choroid plexus in chick embryo

MafB is required for development of the hindbrain choroid plexus

The choroid plexus (ChP) is a non-neural epithelial tissue that produces cerebrospinal fluid (CSF). The ChP differentiates from the roof plate, a dorsal midline structure of the neural tube. However, molecular mechanisms underlying ChP development are poorly understood compared to neural development. MafB is a bZip transcription factor that is known to be expressed in the roof plate. Here we investigated the role of MafB in embryonic development of the hindbrain ChP (hChP) using Mafb-deficient mice. Immunohistochemical analyses revealed that MafB is expressed in the roof plate and early hChP epithelial cells but its expression disappears at a later embryonic stage. We also found that the Mafb-deficient hChP exhibits delayed differentiation and results in hypoplasia compared to the wild-type hChP. Furthermore, the Mafb-deficient hChP exhibits increased apoptotic cell death and decreased proliferating cells at E12.5, an early stage of hChP development. Collectively, our findings reveal that MafB play an important role in promoting hChP development during embryogenesis.

A Distal to Proximal Gradient of Human Choroid Plexus Development, with Antagonistic Expression of Glut1 and AQP1 in Mature Cells vs. Calbindin and PCNA in Proliferative Cells.

The choroid plexuses (ChP) are highly vascularized tissues suspended from each of the cerebral ventricles. Their main function is to secret cerebrospinal fluid (CSF) that fills the ventricles and the subarachnoid spaces, forming a crucial system for the development and maintenance of the CNS. However, despite the essential role of the ChP–CSF system to regulate the CNS in a global manner, it still remains one of the most understudied areas in neurobiology. Here we define by immunohistochemistry the expression of different proteins involved in the maturation and functionality of the ChP from the late embryological period to maturity. We found an opposite gradient of expression between aquaporin 1 (AQP1) and glucose transporter 1 (Glut 1) that define functional maturation in the ChP periphery, and proliferating cell nuclear antigen (PCNA) and calbindin (CB), present in the ChP root zone with proliferative activity. We conclude that the maturation of the ChP matures from distal to proximal, starting in the areas nearest to the cortex, expressing in the distal, mature areas AQP1 and Glut1 (related to ChP functionality to support cortex development), and in the proximal immature areas (ChP root) CB and PCNA related to progenitor activity and proliferation.

The neural milieu of the developing choroid plexus: neural stem cells, neurons and innervation.

The choroid plexus produces cerebrospinal fluid and plays an important role in brain homeostasis both pre and postnatally. In vitro studies have suggested that cells from adult choroid plexus have stem/progenitor cell-like properties. Our initial aim was to investigate whether such a cell population is present in vivo during development of the choroid plexus, focusing mainly on the chick choroid plexus. Cells expressing neural markers were indeed present in the choroid plexus of chick and also those of rodent and human embryos, both within their epithelium and mesenchyme. ß3-tubulin-positive cells with neuronal morphology could be detected as early as at E8 in chick choroid plexus and their morphological complexity increased with development. Whole mount immunochemistry demonstrated the presence of neurons throughout choroid plexus development and they appeared to be mainly catecholaminergic, as indicated by tyrosine-hydroxylase reactivity. The presence of cells co-labeling for BrdU and the neuroblast marker, doublecortin, in organotypic choroid plexus cultures supported the hypothesis that neurogenesis can occur from neural precursors within the developing choroid plexus. Furthermore, we found that extrinsic innervation is present in the developing choroid plexus, unlike previously suggested. Altogether, our data are consistent with the presence of neural progenitors within the choroid plexus, suggest that at least some of the choroid plexus neurons are born locally, and show for the first time that choroid plexus innervation occurs prenatally. Hence, we propose the existence of a complex neural regulatory network within the developing choroid plexus that may play a crucial role in modulating its function during development as well as throughout life.

Development of the choroid plexus and blood-CSF barrier.

Well-known as one of the main sources of cerebrospinal fluid (CSF), the choroid plexuses have been, and still remain, a relatively understudied tissue in neuroscience. The choroid plexus and CSF (along with the blood-brain barrier proper) are recognized to provide a robust protective effort for the brain: a physical barrier to impede entrance of toxic metabolites to the brain; a “biochemical” barrier that facilitates removal of moieties that circumvent this physical barrier; and buoyant physical protection by CSF itself. In addition, the choroid plexus-CSF system has been shown to be integral for normal brain development, central nervous system (CNS) homeostasis, and repair after disease and trauma. It has been suggested to provide a stem-cell like repository for neuronal and astrocyte glial cell progenitors. By far, the most widely recognized choroid plexus role is as the site of the blood-CSF barrier, controller of the internal CNS microenvironment. Mechanisms involved combine structural diffusion restraint from tight junctions between plexus epithelial cells (physical barrier) and specific exchange mechanisms across the interface (enzymatic barrier). The current hypothesis states that early in development this interface is functional and more specific than in the adult, with differences historically termed as “immaturity” actually correctly reflecting developmental specialization. The advanced knowledge of the choroid plexus-CSF system proves itself imperative to understand a range of neurological diseases, from those caused by plexus or CSF drainage dysfunction (e.g., hydrocephalus) to more complicated late-stage diseases (e.g., Alzheimer's) and failure of CNS regeneration. This review will focus on choroid plexus development, outlining how early specializations may be exploited clinically.

Functional and genetic analysis of choroid plexus development in zebrafish.

The choroid plexus, an epithelial-based structure localized in the brain ventricle, is the major component of the blood-cerebrospinal fluid barrier. The choroid plexus produces the cerebrospinal fluid and regulates the components of the cerebrospinal fluid. Abnormal choroid plexus function is associated with neurodegenerative diseases, tumor formation in the choroid plexus epithelium, and hydrocephaly. In this study, we used zebrafish (Danio rerio) as a model system to understand the genetic components of choroid plexus development. We generated an enhancer trap line, Et(cp:EGFP)sj2, that expresses enhanced green fluorescent protein (EGFP) in the choroid plexus epithelium. Using immunohistochemistry and fluorescent tracers, we demonstrated that the zebrafish choroid plexus possesses brain barrier properties such as tight junctions and transporter activity. Thus, we have established zebrafish as a functionally relevant model to study choroid plexus development. Using an unbiased approach, we performed a forward genetic dissection of the choroid plexus to identify genes essential for its formation and function. Using Et(cp:EGFP)sj2, we isolated 10 recessive mutant lines with choroid plexus abnormalities, which were grouped into five classes based on GFP intensity, epithelial localization, and overall choroid plexus morphology. We also mapped the mutation for two mutant lines to chromosomes 4 and 21, respectively. The mutants generated in this study can be used to elucidate specific genes and signaling pathways essential for choroid plexus development, function, and/or maintenance and will provide important insights into how these genetic mutations contribute to disease.

The choroid plexuses and their impact on developmental neurogenesis.

During brain development the neural stem cells are regulated by both intrinsic and extrinsic sources. One site of origin of extrinsic regulation is the developing choroid plexuses, primely situated inside the cerebral ventricles. The choroid plexuses are very active in terms of both secretion and barrier function as soon as they appear during development and control the production and contents of cerebrospinal fluid (CSF). This suggests that regulated secretion of signaling molecules from the choroid plexuses into CSF can regulate neural stem cell behavior (as they are in direct contact with CSF) and thereby neurogenesis and brain development. Here, choroid plexus development, particularly with regards to molecular regulation and specification, is reviewed. This is followed by a review and discussion of the role of the developing choroid plexuses in brain development. In particular, recent evidence suggests a region-specific reciprocal regulation between choroid plexuses and the neural stem cells. This is accomplished by site-specific secretion of signaling molecules from the different choroid plexuses into CSF, as well as brain region specific competence of the neural stem cells to respond to the signaling molecules present in CSF. In conclusion, although in its infancy, the field of choroid plexus regulation of neurogenesis has already and will likely continue to shed new light on our understanding of the control and fine-tuning of overall brain development.

Versican is a critical extracellular matrix protein in lung and brain during embryonic development in the mouse (1046.9)

Versican, a chondroitin sulfate proteoglycan and a component of the extracellular matrix, is associated with fatal cardiac malformations if absent during embryonic development and is increased with injury in adult tissues. Changes in versican gene expression and accumulation have not been examined comprehensively in the lung and brain during mid to late gestation in the mouse. The objective of this work was to determine if versican accumulation and degradation change during lung and brain embryogenesis. Quantitative PCR and immunohistochemistry were used to measure versican expression, accumulation and degradation in the lung, brain, heart and liver of C57Bl/6 mice for five consecutive days during embryonic development. Consistent with previous reports, we found that total versican gene expression was higher in embryonic tissues when compared to adults and the highest total versican gene expression in embryos occurred at embryonic day 13.5 (E13.5). At E15.5, versican gene expression was lowest in the liver, highest in the brain, and intermediate in the heart and lung. There was differential expression of versican isoforms, with V0, V1, and V2 isoforms increased in the brain but only V0 and V1 isoforms increased in the lung. Versican expression in the lung peaked at E13.5, at which time there was strong versican immunoreactivity in the interstitium and airway basement membrane but little accumulation in airway epithelial cells. In the brain, immunoreactivity was strongest in the roof of the hind brain and neopallial cortex and in the deeper white matter tracts, and at later gestational ages there was little accumulation in the developing choroid plexus and periventricular gray matter. During embryonic development of the lung and brain, we found areas of versican expression that colocalized with the expression of DPEAAE, a versican degradation product. These results suggest that versican plays an important role in embryogenesis of the lung and brain.Grant Funding Source: Supported by HL098067, RR030249

Redox-Responsive Mesoporous Silica Nanoparticles: A Physiologically Sensitive Codelivery Vehicle for siRNA and Doxorubicin

Efficient siRNA/drug codelivery carriers can offer great promises to cancer treatment on account of synergistic effect provided from cancer-associated gene and anticancer drugs. In this work, a redox-responsive drug/siRNA codelivery vehicle based on mesoporous silica nanoparticles was fabricated to simultaneously deliver siRNA and doxorubicin (Dox) in vitro and in vivo. The nanoparticle surface was functionalized with the adamantane (AD) units. Formation of stable host-guest complex between disulfide bond linked-AD and ethylenediamine-modified β-cyclodextrin is capable of fully blocking drugs inside the nanopores, while amino groups can complex with siRNA via electrostatic interaction. Relatively high concentration of glutathione in biophysical environment provides natural reducing agent to trigger drug/siRNA release by cleaving pre-introduced disulfide bonds. B-cell lymphoma 2 (Bcl-2) siRNA was codelivered to silence Bcl-2 protein expression in HeLa cells, resulting in enhanced chemotherapy efficacy in vitro. In vivo delivery experiment carried out in transgenic zebrafish larvae indicates that the delivery of Dox inhibits the development of choroid plexus in a dose-dependent manner, leading to successful decrease of green fluorescence protein transcription in choroid plexus. Reduction of liver tumor was also demonstrated after injection of Dox-loaded nanoparticles. We successfully demonstrated that functional nanoparticles could serve as an efficient carrier for the delivery of Bcl-2 siRNA and Dox in HeLa cells and in transgenic zebrafish larvae, leading to enhanced therapeutic efficacy. Enhanced cytotoxicity caused by simultaneous delivery of Bcl-2 siRNA and Dox was observed in HeLa cells. Drug-loaded nanoparticles were internalized in vivo, inhibiting the development of choroid plexus and the progression of liver tumor.