YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers

The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.     

Studying Human Brain Inflammation in Leptomeningeal and Choroid Plexus Explant Cultures

The meninges (dura, pia and arachnoid) are critical membranes encasing and protecting the brain within the skull. The leptomeninges, which comprise the arachnoid and pia, have many functions beyond brain protection including roles in neurogenesis, fibrotic scar formation and brain inflammation. Similarly, the choroid plexus plays important roles in normal brain function but is also involved in brain inflammation. We have begun studying the role of human leptomeninges and choroid plexus in brain inflammation and leptomeninges in fibrotic scar formation, using human brain derived explant cultures. To study the composition of the cells generated in these explants we undertook immunocytochemical characterisation. Cells, mainly pericytes and meningeal macrophages, emerge from leptomeningeal explants (LME’s) and respond to inflammatory mediators by producing inflammatory molecules. LME-derived cells also respond to mechanical injury and cytokines, providing an in vitro human brain model of fibrotic scar formation. Choroid plexus explants (CPE’s) generate epithelial cells, pericytes and microglia/macrophages. CPE-derived cells also respond to inflammatory mediators. LME and CPE explants survive and generate cells for many months in vitro and provide a remarkable opportunity to study basic mechanisms of human brain inflammation and fibrosis and to test human-active anti-inflammatory and anti-scarring treatments.     

BMP signaling is required locally to pattern the dorsal telencephalic midline

BMPs have been proposed to pattern the medial-lateral axis of the telencephalon in a concentration-dependent manner, thus helping to subdivide the embryonic telencephalon into distinct forebrain regions. Using a CRE/loxP genetic approach, we tested this hypothesis by disrupting the Bmpr1a gene in the telencephalon. In mutants, BMP signaling was compromised throughout the dorsal telencephalon, but only the most dorsalmedial derivative, the choroid plexus, failed to be specified or differentiate. Choroid plexus precursors remained proliferative and did not adopt the fate of their lateral telencephalic neighbors. These results demonstrate that BMP signaling is required for the formation of the most dorsal telencephalic derivative, the choroid plexus, and that BMP signaling plays an essential role in locally patterning the dorsal midline. Our data fail to support a more global, concentration-dependent role in specifying telencephalic cell fates.     

The progenitor cells of the embryonic telencephalon and the neonatal anterior subventricular zone differentially regulate their cell cycle

For the last 10 years our laboratory has been studying the proliferation, migration and differentiation of neuronal progenitor cells located in the anterior part of the postnatal forebrain subventricular zone (SVZa). SVZa-derived cells possess a number of proliferative characteristics that distinguish them from the other progenitor cells in the central nervous system. This review summarizes our recent findings, in which we compared the pattern of cell cycle inhibitory proteins expressed by the neonatal SVZa to that of telencephalic ventricular zone cells.     

Sonic hedgehog is required for vascular outgrowth in the hindbrain choroid plexus

Critical to the exchange and metabolic functions served by tissues like brain choroid plexi and lung is the coherent development of an epithelial sheet of large surface area in tight apposition to an extensive vascular bed. Here, we present functional experiments in the mouse demonstrating that Sonic hedgehog (Shh) produced by hindbrain choroid plexus epithelium induces the extensive vascular outgrowths and vascular surface area fundamental to choroid plexus functions, but does not induce the more specialized endothelial cell features of fenestrations and bore size. Our findings indicate that these Shh-dependent vascular elaborations occur even in the presence of Vegf and other established angiogenic factors, suggesting either that the levels of these factors are inadequate in the absence of Shh or that a different set of factors may be more essential to choroid plexus outgrowth. Transducing the Shh signal is a perivascular cell—the pericyte—rather than the more integral vascular endothelial cell itself. Moreover, our findings suggest that hindbrain choroid plexus endothelial cells, as compared to other vascular endothelial cells, are more dependent upon pericytes for instruction. Thus, in addition to Shh acting on the progenitor pool for choroid plexus epithelial cells, as previously shown, it also acts on choroid plexus pericytes, and together serves the important role of coordinating the development of two disparate yet functionally dependent structures—the choroid plexus vasculature and its ensheathing epithelium.     

Knockdown of YKL-40 inhibits angiogenesis through regulation of VEGF/VEGFR2 and ERK1/2 signaling in endometrial cancer

Studies have demonstrated that small interfering RNA (siRNA) targeting YKL-40 (siYKL-40) inhibits the proliferation, migration, invasion, and induces antiapoptotic abilities of endometrial cancer (EC) HEC-1A cells. However, its effect on angiogenesis is unclear. The present study aimed to investigate the role of YKL-40 in endometrial cancer and the related molecular mechanisms. YKL-40 was knocked down by transfection with siYKL-40 and the effects on angiogenesis, cell viability, and signaling pathways were investigated. The results showed that siYKL-40 inhibited VEGFA levels and tube formation in endothelial cells. Additionally, inhibition of YKL-40 decreased the expression levels of vascular endothelial growth factor (VEGF), phosphorylated vascular endothelial growth factor receptor 2 (pVEGFR2), and phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2). Furthermore, a nude mice xenograft model of EC showed that siYKL-40 inhibited tumor growth. Inhibition of YKL-40 led to suppression of angiogenesis and reduction of microvessel density through VEGF/VEGFR2 and ERK1/2 signaling in endometrial cancer cells. Taken together, this study demonstrated novel molecular mechanisms for role of YKL-40 in EC.     

Choroid plexus and paraphysis in lower vertebrates

Cytoarchitecture of the choroid plexus of the third ventricle and the paraphysis was investigated in some lower vertebrates to compare the histologic characteristics of these organs. Both epithelia are similar in appearance in the same class. Minor microscopic variations exist in the different classes of vertebrates, but do not provide a fundamental distinction between the two organs. The epithelia, moreover, have similar staining properties, contain mucicarmine- and PAS-reactive materials, and are derived from a common neuroepithelium. Tubules are identified in the choroid plexus and in the paraphysis; all are similarly formed by simple folding of epithelium on the surface into the stroma. The paraphyses in all vertebrates studied contain villi similar to those seen in the choroid plexus. Cilia are identified in both choroidal and paraphyseal epithelia, and are not an indication of degree of epithelial differentiation. Many types of epithelium are noted in both organs during histologic differentiation as well as in the mature stage. Functionally, the choroid plexus is active in both secretion and absorption. Accumulation of particulate material within the epithelial cytoplasm may indicate phagocytic as well as absorptive activity of cells. Based on a common neuroepithelial origin and similar histochemical properties, we conclude that the paraphysis is a modified choroid plexus. The velum transversum is an arbitrary boundary between diencephalon and telencephalon, and is itself formed of choroid plexus. The medial telencephalic ventricle is the rostral portion of the third ventricle. All neuroepithelial infoldings at the rostral end of the diencephalic roof including the velum transversum are intraventricular choroid plexuses; the neuroepithelial outpouchings in this region are the extraventricular choroid plexuses (paraphysis) of the diencephalon.     

YKL-40 protein expression in the early developing human musculoskeletal system.

YKL-40 is a growth factor for chondrocytes and fibroblasts. The aim was to evaluate YKL-40 expression in the musculoskeletal system during early human development. We studied sections from 15 human embryos [weeks 5.5-8; 7- to 31-mm crown-rump length (CRL)] and 68 fetuses (weeks 9-14; 33- to 105-mm CRL) for YKL-40 protein expression by immunohistochemistry. YKL-40 mRNA expression was evaluated in two human embryos (days 41 and 51). Initially YKL-40 is expressed in all germ layers: ecto-, meso-, and endoderm. YKL-40 mRNA and protein expression are found in tissues of the ecto-, meso-, and endoderm, and YKL-40 protein expression is present during development of cartilage, bone, joints, and muscles. At the cellular level, YKL-40 protein expression is high in tissues characterized by rapid proliferation, marked differentiation, and undergoing morphogenetic changes. Examples of rapid cell proliferation include the chondrogenic inner layer of perichondrium and the osteogenic inner layer of periosteum. Differences in YKL-40 expression during differentiation are found in the chondrogenic and osteogenic cell lineages. The initial shaping of cartilage and bone models and joints is concomitant with a strong outline of YKL-40-positive cells. This indicates that YKL-40 is associated with cell proliferation, differentiation, and tissue morphogenesis during development of the human musculoskeletal system.     

Inhibitory activity of YKL-40 in mammary epithelial cell differentiation and polarization induced by lactogenic hormones: a role in mammary tissue involution

We previously reported that a secreted glycoprotein YKL-40 acts as an angiogenic factor to promote breast cancer angiogenesis. However, its functional role in normal mammary gland development is poorly understood. Here we investigated its biophysiological activity in mammary epithelial development and mammary tissue morphogenesis. YKL-40 was expressed exclusively by ductal epithelial cells of parous and non-parous mammary tissue, but was dramatically up-regulated at the beginning of involution. To mimic ductal development and explore activity of elevated YKL-40 during mammary tissue regression in vivo, we grew a mammary epithelial cell line 76N MECs in a 3-D Matrigel system in the presence of lactogenic hormones including prolactin, hydrocortisone, and insulin. Treatment of 76N MECs with recombinant YKL-40 significantly inhibited acinar formation, luminal polarization, and secretion. YKL-40 also suppressed expression of E-cadherin but increased MMP-9 and cell motility, the crucial mechanisms that mediate mammary tissue remodeling during involution. In addition, engineering of 76N MECs with YKL-40 gene to express ectopic YKL-40 recapitulated the same activities as recombinant YKL-40 in the inhibition of cell differentiation. These results suggest that YKL-40-mediated inhibition of cell differentiation and polarization in the presence of lactogenic hormones may represent its important function during mammary tissue involution. Identification of this biophysiological property will enhance our understanding of its pathologic role in the later stage of breast cancer that is developed from poorly differentiated and highly invasive cells.     

Choroid plexus recovery after transient forebrain ischemia: role of growth factors and other repair mechanisms

1. Transient forebrain ischemia in adult rats, induced by 10 min of bilateral carotid occlusion and an arterial hypotension of 40 mmHg, caused substantial damage not only to CA-1 neurons in hippocampus but also to epithelial cells in lateral ventricle choroid plexus.2. When transient forebrain ischemia was followed by reperfusion (recovery) intervals of 0 to 12 hr, there was moderate to severe damage to many frond regions of the choroidal epithelium. In some areas, epithelial debris was sloughed into cerebrospinal fluid (CSF). Although some epithelial cells were disrupted and necrotic, their neighbors exhibited normal morphology. This patchy response to ischemia was probably due to regional differences in reperfusion or cellular metabolism.3. Between 12 and 24 hr postischemia, there was marked restoration of the Na⁺, K⁺, water content, and ultrastructure of the choroid plexus epithelium. Since there was no microscopical evidence for mitosis, we postulate that healthy epithelial cells either were compressed together on the villus or migrated from the choroid plexus stalk to more distal regions, in order to fill in gaps along the basal lamina caused by necrotic epithelial cell disintegration.4. Epithelial cells of mammalian choroid plexus synthesize and secrete many growth factors and other peptides that are of trophic benefit following injury to regions of the cerebroventricular system. For example, several growth factors are upregulated in choroid plexus after ischemic and traumatic insults to the central nervous system.5. The presence of numerous types of growth factor receptors in choroid plexus allows growth factor mediation of recovery processes by autocrine and paracrine mechanisms.6. The capability of choroid plexus after acute ischemia to recover its barrier and CSF formation functions is an important factor in stabilizing brain fluid balance.7. Moreover, growth factors secreted by choroid plexus into CSF are distributed by diffusion and convection into brain tissue near the ventricular system, e.g., hippocampus. By this endocrine-like mechanism, growth factors are conveyed throughout the choroid plexus–CSF–brain nexus and can consequently promote repair of ischemia-damaged tissue in the ventricular wall and underlying brain.     

Role of YKL-40 in the angiogenesis, radioresistance, and progression of glioblastoma

Glioblastoma is one of the most fatal cancers, characterized by a strong vascularized phenotype. YKL-40, a secreted glycoprotein, is overexpressed in patients with glioblastomas and has potential as a novel tumor biomarker. The molecular mechanisms of YKL-40 in glioblastoma development, however, are poorly understood. Here, we aimed to elucidate the role YKL-40 plays in the regulation of VEGF expression, tumor angiogenesis, and radioresistance. YKL-40 up-regulated VEGF expression in glioblastoma cell line U87, and both YKL-40 and VEGF synergistically promote endothelial cell angiogenesis. Interestingly, long term inhibition of VEGF up-regulated YKL-40. YKL-40 induced coordination of membrane receptor syndecan-1 and integrin αvβ5, and triggered a signaling cascade through FAK(397) to ERK-1 and ERK-2, leading to elevated VEGF and enhanced angiogenesis. In addition, γ-irradiation of U87 cells increased YKL-40 expression that protects cell death through AKT activation and also enhances endothelial cell angiogenesis. Blockade of YKL-40 activity or expression decreased tumor growth, angiogenesis, and metastasis in xenografted animals. Immunohistochemical analysis of human glioblastomas revealed a correlation between YKL-40, VEGF, and patient survival. These findings have shed light on the mechanisms by which YKL-40 promotes tumor angiogenesis and malignancy, and thus provide a therapeutic target for tumor treatment.     

Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline

The choroid plexus in the brain is unique because it is a non-neural secretory tissue. It secretes the cerebrospinal fluid and functions as a blood-brain barrier, but the precise mechanism of specification of this non-neural tissue has not yet been determined. Using mouse embryos and lineage-tracing analysis, we found that the prospective choroid plexus region initially gives rise to Cajal-Retzius cells, specialized neurons that guide neuronal migration. Inactivation of the bHLH repressor genes Hes1, Hes3 and Hes5 upregulated expression of the proneural gene neurogenin 2 (Ngn2) and prematurely depleted Bmp-expressing progenitor cells, leading to enhanced formation of Cajal-Retzius cells and complete loss of choroid plexus epithelial cells. Overexpression of Ngn2 had similar effects. These data indicate that Hes genes promote specification of the fate of choroid plexus epithelial cells rather than the fate of Cajal-Retzius cells by antagonizing Ngn2 in the dorsal telencephalic midline region, and thus this study has identified a novel role for bHLH genes in the process of deciding which cells will have a non-neural versus a neural fate.     

Involvement of metalloprotease-2 in the development of human brain microvessels

The involvement of the metalloprotease-2 (MMP-2) in vessel development was investigated in the human telencephalon by double immunoreactions with antibodies to the enzyme, latent (proMMP-2) and active (aMMP-2) forms, and an antibody against collagen type IV, a constitutive component of the extracellular matrix (ECM) of the vessel basal lamina. MMP-2 is expressed in both 12- and 18-week telencephalic vessels, the proenzyme being mainly localised in endothelial cells and the active form prevailing in -actin-reactive periendothelial cells identified as pericytes. Endothelial cells intensely positive for aMMP-2 were revealed in some microvessels and appeared locally associated with discontinuities of the collagen basal lamina. No detectable expression of MMP-2 was observed in perivascular glial processes revealed by vimentin/glial fibrillary acidic protein immunostainings. Double immunoreactions performed to further investigate telencephalon angiogenesis have demonstrated that both the endothelial cells and pericytes strongly express vascular endothelial growth factor (VEGF). Taken together, the results indicate that MMP-2 is largely involved in human brain angiogenesis and suggest that endothelial cells and pericytes tightly interplay in both angiogenesis mechanisms, by ECM proteolysis, and angiogenesis regulation, by local (autocrine/juxtacrine) VEGF action.Francesco Girolamo and Daniela Virgintino contributed equally to this work     

The Vascularization of the Anuran Brain Diencephalon and Choroid Plexus: A scanning electron microscopical study of vascular corrosion casts

Albrecht, U., Lametschwandtner, A., Adam, H. 1979. The vascularization of the anuran brain. Diencephalon and choroid plexus. A scanning electron microscopical study of vascular corrosion casts. (Department of Zoology, University of Saulzburg, Austria.) — Acta zool. (Stockh.) 61(4): 203–220. The vascularization of the diencephalon (with choroid plexus of the third ventricle, epithalamus and pineal region, thalamus and hypothalamus) of the toad, Bufo bufo (L.) has been studied by means of scanning electron microscopy of vascular corrosion casts. To localize angioarchitectonic patterns of distinct diencephalic regions the authors refer to critical point dried specimens and to histological sections. In the choroid plexus a supply via one choroid artery, which arises from the posterior telencephalic artery, was found. Its strict dichotomous branching is pointed out. In generally a similar vascular pattern like that in the choroid plexus of the fourth ventricle has to be reported. Furthermore the epithalamic region with the epiphysial area was under investigation. No special angioarchitecture of the epiphysis was found. There are also no prominent vascular connections with thalamic or hypothalamic regions. The thalamic region is supplied by branches of the posterior telencephalic artery as well as by branches of the preoptic artery. Epithalamic and thalamic regions are drained via the posterior diencephalic vein. Special attention was also paid to the preoptic, the chiasmatic and the retrochiasmatic area. No special vascular connections, however, were found.     

Morphological Ground of Formation of the Brain Choroid Plexus Blood-Cerebrospinal Fluid Barrier in Human Prenatal Ontogenesis

Using light and electron microscopy and immunocytochemistry methods, structural organization of the formed blood-cerebrospinal fluid barrier (BCSFB) of the human brain choroid plexus in embryos of 6–9 weeks of development was studied. The main structures peculiar to the mature BCSFB have been established to appear with formation of the choroid plexus at the end of the 2nd month of the human intrauterine development. Fenestrae in the choroid plexus capillary endothelium are revealed since the 9th week of prenatal development. Characteristic of the human embryonic BCSFB are a poor development of the plexus capillary basal membrane, scanty pericytes, a high activity of interstitial macrophages, which suggests the barrier immaturity. A significant amount of cytoplasmic glycogen inclusions revealed in plexus epitheliocytes seems to be due to peculiar trophic requirements of developing brain cells under conditions of an insufficient development of the local blood supply.     

A novel role for the choroid plexus in BMP-mediated inhibition of differentiation of cerebellar neural progenitors

Cerebellar granule cells, the most abundant neurons in the mammalian brain, arise in the rhombic lip located at the roof of the brain's fourth ventricle. Bordering the rhombic lip is the choroid plexus, a non-neuronal structure, composed of blood vessels enveloped by epithelial cells. Here, we show a striking decrease in neural differentiation of rhombic lip-derived cells, which failed to extend neuritic processes and attenuate Math1 promoter activity, when co-cultured with choroid plexus cells. Moreover, a blocking antibody against BMP7, a morphogenetic protein expressed in the choroid plexus, blocked the inhibitory effect of the choroid plexus, whereas purified BMP7 mimicked this effect, demonstrating causal involvement of BMP. On the other hand, the BMP antagonist NBL1 promoted neurogenesis in rhombic lip cultures from Math1 null mice displaying arrested differentiation. Our data indicate that besides its secretory and barrier functions, the choroid plexus has a novel role in attenuating the differentiation of adjacent neural progenitors.     

Penetration and differentiation of cephalic neural crest‐derived cells in the developing mouse telencephalon

Neural crest (NC) cells originate from the neural folds and migrate into the various embryonic regions where they differentiate into multiple cell types. A population of cephalic neural crest‐derived cells (NCDCs) penetrates back into the developing forebrain to differentiate into microvascular pericytes, but little is known about when and how cephalic NCDCs invade the telencephalon and differentiate into pericytes. Using a transgenic mouse line in which NCDCs are genetically labeled with enhanced green fluorescent protein (EGFP), we observed that NCDCs started to invade the telencephalon together with endothelial cells from embryonic day (E) 9.5. A majority of NCDCs located in the telencephalon expressed pericyte markers, that is, PDGFRβ and NG2, and differentiated into pericytes around E11.5. Surprisingly, many of the NC‐derived pericytes express p75, an undifferentiated NCDC marker at E11.5, as well as NCDCs in the mesenchyme. At the same time, a minor population of NCDCs that located separately from blood vessels in the telencephalon were NG2‐negative and some of these NCDCs also expressed p75. Proliferation and differentiation of pericytes appeared to occur in a specific mesenchymal region where blood vessels penetrated into the telencephalon. These results indicate that (i) NCDCs penetrate back into the telencephalon in parallel with angiogenesis, (ii) many NC‐derived pericytes may be still in pre‐mature states even though after differentiation into pericytes in the early developing stages, (iii) a small minority of NCDCs may retain undifferentiated states in the developing telencephalon, and (iv) a majority of NCDCs proliferate and differentiate into pericytes in the mesenchyme around the telencephalon.     

Gli3 is required for Emx gene expression during dorsal telencephalon development.

Dentate gyrus and hippocampus as centers for spatial learning, memory and emotional behaviour have been the focus of much interest in recent years. The molecular information on its development, however, has been relatively poor. To date, only Emx genes were known to be required for dorsal telencephalon development. Here, we report on forebrain development in the extra toes (Xt(J)) mouse mutant which carries a null mutation of the Gli3 gene. This defect leads to a failure to establish the dorsal di-telencephalic junction and finally results in a severe size reduction of the neocortex. In addition, Xt(J)/Xt(J) mice show absence of the hippocampus (Ammon's horn plus dentate gyrus) and the choroid plexus in the lateral ventricle. The medial wall of the telencephalon, which gives rise to these structures, fails to invaginate during embryonic development. On a molecular level, disruption of dorsal telencephalon development in Xt(J)/Xt(J) embryos correlates with a loss of Emx1 and Emx2 expression. Furthermore, the expression of Fgf8 and Bmp4 in the dorsal midline of the telencephalon is altered. However, expression of Shh, which is negatively regulated by Gli3 in the spinal cord, is not affected in the Xt(J)/Xt(J) forebrain. This study therefore implicates Gli3 as a key regulator for the development of the dorsal telencephalon and implies Gli3 to be upstream of Emx genes in a genetic cascade controlling dorsal telencephalic development.     

A critical role for pannexin-1 in activation of innate immune cells of the choroid plexus

Epiplexus cells are a population of innate immune cells in the choroid plexus of the brain ventricles. They are thought to contribute to the immune component of the blood-cerebrospinal-fluid-barrier (BCSFB). Here we have developed a novel technique for studying epiplexus cells in acutely isolated, live and intact choroid plexus. We show that epiplexus cells are potently activated by exogenous ATP, increasing their motility within the tissue. This ATP-induced chemokinesis required activation of pannexin-1 channels, which are expressed by the epithelial cells of the choroid plexus and not the epiplexus cells themselves. Furthermore, ATP acts at least in part through the P2X4 ionotropic purinergic receptor. Thus, the resident immune cells of the choroid plexus appear to be in communication with the epithelial cells through pannexin-1 channels.