Development and fibronectin signaling requirements of the zebrafish interrenal vessel

Background

The early morphogenetic steps of zebrafish interrenal tissue, the teleostean counterpart of the mammalian adrenal gland, are modulated by the peri-interrenal angioblasts and blood vessels. While an organized distribution of intra-adrenal vessels and extracellular matrix is essential for the fetal adrenal cortex remodeling, whether and how an intra-interrenal buildup of vasculature and extracellular matrix forms and functions during interrenal organogenesis in teleosts remains unclear.

Methodology and Principal Findings

We characterized the process of interrenal gland vascularization by identifying the interrenal vessel (IRV); which develops from the axial artery through angiogenesis and is associated with highly enriched Fibronectin (Fn) accumulation at its microenvironment. The loss of Fn1 by either antisense morpholino (MO) knockdown or genetic mutation inhibited endothelial invasion and migration of the steroidogenic tissue. The accumulation of peri-IRV Fn requires Integrin α5 (Itga5), with its knockdown leading to interrenal and IRV morphologies phenocopying those in the fn1 morphant and mutant. fn1b, another known fn gene in zebrafish, is however not involved in the IRV formation. The distribution pattern of peri-IRV Fn could be modulated by the blood flow, while a lack of which altered angiogenic direction of the IRV as well as its ability to integrate with the steroidogenic tissue. The administration of Fn antagonist through microangiography exerted reducing effects on both interrenal vessel angiogenesis and steroidogenic cell migration.

Conclusions and Significance

This work is the first to identify the zebrafish IRV and to characterize how its integration into the developing interrenal gland requires the Fn-enriched microenvironment, which leads to the possibility of using the IRV formation as a platform for exploring organ-specific angiogenesis. In the context of other developmental endocrinology studies, our results indicate a highly dynamic interrenal-vessel interaction immediately before the onset of stress response in the zebrafish embryo.     

In vivo analysis of choroid plexus morphogenesis in zebrafish

Background

The choroid plexus (ChP), a component of the blood-brain barrier (BBB), produces the cerebrospinal fluid (CSF) and as a result plays a role in (i) protecting and nurturing the brain as well as (ii) in coordinating neuronal migration during neurodevelopment. Until now ChP development was not analyzed in living vertebrates due to technical problems.

Methodology/Principal Findings

We have analyzed the formation of the fourth ventricle ChP of zebrafish in the GFP-tagged enhancer trap transgenic line SqET33-E20 (Gateways) by a combination of in vivo imaging, histology and mutant analysis. This process includes the formation of the tela choroidea (TC), the recruitment of cells from rhombic lips and, finally, the coalescence of TC resulting in formation of ChP. In Notch-deficient mib mutants the first phase of this process is affected with premature GFP expression, deficient cell recruitment into TC and abnormal patterning of ChP. In Hedgehog-deficient smu mutants the second phase of the ChP morphogenesis lacks cell recruitment and TC cells undergo apoptosis.

Conclusions/Significance

This study is the first to demonstrate the formation of ChP in vivo revealing a role of Notch and Hedgehog signalling pathways during different developmental phases of this process.     

Asymmetric localization of Notch2 on the microvillous surface in choroid plexus epithelial cells

Notch family molecules are transmembrane receptors that play various roles in contact-dependent cell–cell interactions in a wide range of organs. In the brain, Notch2, but not the other members of Notch, is expressed in the choroid plexus at an exceptionally high level. We immunohistochemically examined the cellular and subcellular localization of Notch2 protein in the choroid plexus using confocal and electron microscopy. Unexpectedly, Notch2 was asymmetrically localized on the microvillous surface of epithelial cells in the choroid plexus of both postnatal and adult rats. This localization pattern of Notch2 suggests its novel and unknown role independent of contact with adjacent cells in the choroid plexus. In organotypic cultures of the choroid plexus, the addition of anti-Notch2 antibody resulted in deformation of microvilli in epithelial cells, which suggests a role of Notch2 in the maintenance of the microvillous structure in choroid plexus epithelial cells.     

Notch signaling regulates endocrine cell specification in the zebrafish anterior pituitary

The vertebrate pituitary gland is a key endocrine control organ that contains six distinct hormone secreting cell types. In this study, we analyzed the role of direct cell-to-cell Delta-Notch signaling in zebrafish anterior pituitary cell type specification. We demonstrate that initial formation of the anterior pituitary placode is independent of Notch signaling. Later however, loss of Notch signaling in mind bomb (mib) mutant embryos or by DAPT treatment leads to increased numbers of lactotropes and loss of corticotropes in the anterior pars distalis (APD), increased number of thyrotropes and loss of somatotrope cell types in the posterior pars distalis (PPD), and fewer melanotropes in the posterior region of the adenohypophysis, the pars intermedia (PI). Conversely, Notch gain of function leads to the opposite result, loss of lactotrope and thyrotrope cell specification, and an increased number of corticotropes, melanotropes, and gonadotropes in the pituitary. Our results suggest that Notch acts on placodal cells, presumably as a permissive signal, to regulate progenitor cell specification to hormone secreting cell types. We propose that Notch mediated lateral inhibition regulates the relative numbers of specified hormone cell types in the three pituitary subdomains.     

Discrete Notch signaling requirements in the specification of hematopoietic stem cells

Hematopoietic stem cells (HSCs) require multiple molecular inputs for proper specification, including activity of the Notch signaling pathway. A requirement for the Notch1 and dispensability of the Notch2 receptor has been demonstrated in mice, but the role of the remaining Notch receptors has not been investigated. Here, we demonstrate that three of the four Notch receptors are independently required for the specification of HSCs in the zebrafish. The orthologues of the murine Notch1 receptor, Notch1a and Notch1b, are each required intrinsically to fate HSCs, just prior to their emergence from aortic hemogenic endothelium. By contrast, the Notch3 receptor is required earlier within the developing somite to regulate HSC emergence in a non-cell-autonomous manner. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld. Collectively, these findings demonstrate for the first time that multiple Notch signaling inputs are required to specify HSCs and that Notch3 performs a novel role within the somite to regulate the neighboring precursors of hemogenic endothelium.     

Notch signaling and Notch signaling modifiers

Originally discovered nearly a century ago, the Notch signaling pathway is critical for virtually all developmental programs and modulates an astounding variety of pathogenic processes. The DSL (Delta, Serrate, LAG-2 family) proteins have long been considered canonical activators of the core Notch pathway. More recently, a wide and expanding network of non-canonical extracellular factors has also been shown to modulate Notch signaling, conferring newly appreciated complexity to this evolutionarily conserved signal transduction system. Here, I review current concepts in Notch signaling, with a focus on work from the last decade elucidating novel extracellular proteins that up- or down-regulate signal potency.     

Cell trafficking through the choroid plexus

The choroid plexus is a multifunctional organ that sits at the interface between the blood and cerebrospinal fluid (CSF). It serves as a gateway for immune cell trafficking into the CSF and is in an excellent position to provide continuous immune surveillance by CD4⁺ T cells, macrophages and dendritic cells and to regulate immune cell trafficking in response to disease and trauma. However, little is known about the mechanisms that control trafficking through this structure. Three cell types within the choroid plexus, in particular, may play prominent roles in controlling the development of immune responses within the nervous system: the epithelial cells, which form the blood-CSF barrier, and resident macrophages and dendritic cells in the stromal matrix. Adhesion molecule and chemokine expression by the epithelial cells allows substantial control over the selection of cells that transmigrate. Macrophages and dendritic cells can present antigen within the choroid plexus and/or transmigrate into the cerebral ventricles to serve a variety of possible immune functions. Studies to better understand the diverse functions of these cells are likely to reveal new insights that foster the development of novel pharmacological and macrophage-based interventions for the control of CNS immune responses.     

Cell trafficking through the choroid plexus

The choroid plexus is a multifunctional organ that sits at the interface between the blood and cerebrospinal fluid (CSF). It serves as a gateway for immune cell trafficking into the CSF and is in an excellent position to provide continuous immune surveillance by CD4⁺ T cells, macrophages and dendritic cells and to regulate immune cell trafficking in response to disease and trauma. However, little is known about the mechanisms that control trafficking through this structure. Three cell types within the choroid plexus, in particular, may play prominent roles in controlling the development of immune responses within the nervous system: the epithelial cells, which form the blood-CSF barrier, and resident macrophages and dendritic cells in the stromal matrix. Adhesion molecule and chemokine expression by the epithelial cells allows substantial control over the selection of cells that transmigrate. Macrophages and dendritic cells can present antigen within the choroid plexus and/or transmigrate into the cerebral ventricles to serve a variety of possible immune functions. Studies to better understand the diverse functions of these cells are likely to reveal new insights that foster the development of novel pharmacological and macrophage-based interventions for the control of CNS immune responses.     

Sodium localization in the choroid plexus

Summary The localization of sodium ion in the cat choroid plexus was studied by use of potassium pyroantimonate. The precipitates formed by the potassium pyroantimonate occur mostly on the plasma membrane in the epithelial cell and occasionally in the perivascular space. The precipitates in the epithelial cell are most numerous at the apical surface, particularly on the microvilli, and least in number at the basal and lateral surfaces. In the endothelial cell, the dense precipitates are situated on the plasma membrane as well as on the limiting membrane of the pinocytotic vesicle. Although the dense precipitates are sometimes situated on the external surface of the plasma membrane of the epithelial cell, most of them are localized on the internal surface of the plasma membrane. A similar localization of the precipitates is to be seen on the plasma membrane of the erythrocyte. When the cerebrospinal fluid/plasma ion ratio and potential gradients across the choroid plexus are considered, the precipitates on the plasma membrane would suggest a localization of sodium needed for the activation of ATPase.

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Zusammenfassung Die Lokalisation des Natriumions im Plexus chorioideus der Katze wurde mit Hilfe von Kaliumpyroantimonat untersucht. Die durch Kaliumpyroantimonat gebildeten Niederschläge treten meistens an der Plasmamembran in den Epithelzellen und gelegentlich im perivaskulären Raum auf. In den Epithelzellen kommen die Niederschläge am zahlreichsten an der apikalen Oberfläche vor, besonders an den Mikrovilli, am geringsten an den basalen und lateralen Oberflächen. In der Endothelzelle liegen die dichten Niederschläge an der Plasmamembran und an der Grenzmembran der Pinozytosebläschen. Einige der dichten Niederschläge befinden sich an der äußeren Oberfläche der Plasmamembran der Epithelzellen, die meisten aber an der inneren Oberfläche der Plasmamembran. Eine ähnliche Lokalisation der Niedersschläge wurde an der Plasmamembran des Erythrozyten festgestellt. Wenn man das Liquor Plasma-Ionenverhältnis und die Potentialgradienten am Plexus chorioideus in Betracht zieht, liegt es nahe, die nachgewiesene Lokalisation des Natriums auf eine Aktivierung von ATPase zu beziehen.

     

Differential requirements for Wnt and Notch signaling in hematopoietic versus thymic niches

All blood cells are derived from multipotent stem cells, the so-called hematopoietic stem cells (HSCs), that in adults reside in the bone marrow. Most types of blood cells also develop there, with the notable exception of T lymphocytes that develop in the thymus. For both HSCs and developing T cells, interactions with the surrounding microenvironment are critical in regulating maintenance, differentiation, apoptosis, and proliferation. Such specialized regulatory microenvironments are referred to as niches and provide both soluble factors as well as cell-cell interactions between niche component cells and blood cells. Two pathways that are critical for early T cell development in the thymic niche are Wnt and Notch signaling. These signals also play important but controversial roles in the HSC niche. Here, we review the differences and similarities between the thymic and hematopoietic niches, with particular focus on Wnt and Notch signals, as well as the latest insights into regulation of these developmentally important pathways.     

Vasopressin V1a receptor signaling in a rat choroid plexus cell line

A new cell line was derived from primary culture of rat choroid plexus (RCP) by immortalization with the TSOri minus adenovirus. The selected clone expressed vasopressin V1a receptors at a density of 64,000 sites per cell, and a K(d) of 7.2 nM. Addition of vasopressin to the RCP cells induced a transient calcium peak comparable to V1a receptor signalling in different expression systems. This [Ca(2+)](i) increase was dose-dependent with an EC(50) of 22 nM vasopressin. Similar [Ca(2+)](i) increase was elicited by addition of serotonin, angiotensin II, endothelin-1, and bradykinin. Heterologous desensitization of V1a receptor was observed in RCP cells exposed to the phorbol ester PMA or following stimulation of other receptors coupled to the phosphoinositide pathway. Positive immunolabelling with Factor VIII, Flt1 and CD 34 antibodies suggests that this new RCP cell line originated from endothelial cells of rat choroid plexus.     

Notch signaling regulates neural precursor allocation and binary neuronal fate decisions in zebrafish

Notch signaling plays a well-described role in regulating the formation of neurons from proliferative neural precursors in vertebrates but whether, as in flies, it also specifies sibling cells for different neuronal fates is not known. Ventral spinal cord precursors called pMN cells produce mostly motoneurons and oligodendrocytes, but recent lineage-marking experiments reveal that they also make astrocytes, ependymal cells and interneurons. Our own clonal analysis of pMN cells in zebrafish showed that some produce a primary motoneuron and KA' interneuron at their final division. We investigated the possibility that Notch signaling regulates a motoneuron-interneuron fate decision using a combination of mutant, transgenic and pharmacological manipulations of Notch activity. We show that continuous absence of Notch activity produces excess primary motoneurons and a deficit of KA' interneurons, whereas transient inactivation preceding neurogenesis results in an excess of both cell types. By contrast, activation of Notch signaling at the neural plate stage produces excess KA' interneurons and a deficit of primary motoneurons. Furthermore, individual pMN cells produce similar kinds of neurons at their final division in mib mutant embryos, which lack Notch signaling. These data provide evidence that, among some postmitotic daughters of pMN cells, Notch promotes KA' interneuron identity and inhibits primary motoneuron fate, raising the possibility that Notch signaling diversifies vertebrate neuron type by mediating similar binary fate decisions.     

Development of transport mechanisms for sulphate and iodide in immature choroid plexus

The time-course of development of sulphate and iodide transport mechanisms in choroid plexus was studied by measuring uptake of [³⁵S]sulphate and [¹²⁵I]iodide from an incubating medium by isolated choroid plexuses of foetal and newborn rabbits and cats. Sulphate uptake by choroid plexus was poorly developed in rabbit foetuses just before term, but highly developed in newborn animals. Iodide uptake was already well developed in the most immature foetuses studied.