Monorail/Foxa2 regulates floorplate differentiation and specification of oligodendrocytes, serotonergic raphe neurones and cranial motoneurones

In this study, we elucidate the roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish. Through cloning of monorail (mol), which we find encodes the transcription factor Foxa2, and phenotypic analysis of mol(-/-) embryos, we show that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol(-/-) mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. Our results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves. Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurones of the raphe nucleus and the trochlear motor nucleus are absent in mol(-/-) embryos, and oculomotor and facial motoneurones ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins

Zebrafish usp39 Mutation Leads to rb1 mRNA Splicing Defect and Pituitary Lineage Expansion

Loss of retinoblastoma (Rb) tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39) mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130), and cdkn1a (p21) expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis

Anaplastic Lymphoma Kinase Is Required for Neurogenesis in the Developing Central Nervous System of Zebrafish

10.1371/journal.pone.0063757PLoS ONE85

Nodal-Dependent Mesendoderm Specification Requires the Combinatorial Activities of FoxH1 and Eomesodermin

Vertebrate mesendoderm specification requires the Nodal signaling pathway and its transcriptional effector FoxH1. However, loss of FoxH1 in several species does not reliably cause the full range of loss-of-Nodal phenotypes, indicating that Nodal signals through additional transcription factors during early development. We investigated the FoxH1-dependent and -independent roles of Nodal signaling during mesendoderm patterning using a novel recessive zebrafish FoxH1 mutation called midway, which produces a C-terminally truncated FoxH1 protein lacking the Smad-interaction domain but retaining DNA–binding capability. Using a combination of gel shift assays, Nodal overexpression experiments, and genetic epistasis analyses, we demonstrate that midway more accurately represents a complete loss of FoxH1-dependent Nodal signaling than the existing zebrafish FoxH1 mutant schmalspur. Maternal-zygotic midway mutants lack notochords, in agreement with FoxH1 loss in other organisms, but retain near wild-type expression of markers of endoderm and various nonaxial mesoderm fates, including paraxial and intermediate mesoderm and blood precursors. We found that the activity of the T-box transcription factor Eomesodermin accounts for specification of these tissues in midway embryos. Inhibition of Eomesodermin in midway mutants severely reduces the specification of these tissues and effectively phenocopies the defects seen upon complete loss of Nodal signaling. Our results indicate that the specific combinations of transcription factors available for signal transduction play critical and separable roles in determining Nodal pathway output during mesendoderm patterning. Our findings also offer novel insights into the co-evolution of the Nodal signaling pathway, the notochord specification program, and the chordate branch of the deuterostome family of animals

Kif1b is essential for mRNA localization in oligodendrocytes and development of myelinated axons

The kinesin motor protein Kif1b has previously been implicated in the axonal transport of mitochondria and synaptic vesicles1,2. More recently kif1b has been linked with susceptibility to Multiple Sclerosis (MS) 3. Here we show that Kif1b is required for the localization of myelin basic protein mRNA to processes of myelinating oligodendrocytes in zebrafish. We observe the ectopic appearance of myelin-like membrane in kif1b mutants, coincident with the ectopic localization of myelin proteins in kif1b mutant oligodendrocyte cell bodies. These observations suggest the hypothesis that oligodendrocytes localize certain mRNA molecules, namely those encoding small basic proteins such as mbp, to prevent aberrant effects of these proteins elsewhere in the cell. We also find that Kif1b is required for outgrowth of some of the longest axons in the peripheral and central nervous systems. Our data demonstrate new functions of kif1b in vivo and provide insights into its possible roles in Multiple Sclerosis

Myelin Proteomics: Molecular Anatomy of an Insulating Sheath

Fast-transmitting vertebrate axons are electrically insulated with multiple layers of nonconductive plasma membrane of glial cell origin, termed myelin. The myelin membrane is dominated by lipids, and its protein composition has historically been viewed to be of very low complexity. In this review, we discuss an updated reference compendium of 342 proteins associated with central nervous system myelin that represents a valuable resource for analyzing myelin biogenesis and white matter homeostasis. Cataloging the myelin proteome has been made possible by technical advances in the separation and mass spectrometric detection of proteins, also referred to as proteomics. This led to the identification of a large number of novel myelin-associated proteins, many of which represent low abundant components involved in catalytic activities, the cytoskeleton, vesicular trafficking, or cell adhesion. By mass spectrometry-based quantification, proteolipid protein and myelin basic protein constitute 17% and 8% of total myelin protein, respectively, suggesting that their abundance was previously overestimated. As the biochemical profile of myelin-associated proteins is highly reproducible, differential proteome analyses can be applied to material isolated from patients or animal models of myelin-related diseases such as multiple sclerosis and leukodystrophies

Zebrafish regenerate full thickness optic nerve myelin after demyelination, but this fails with increasing age

INTRODUCTION: In the human demyelinating central nervous system (CNS) disease multiple sclerosis, remyelination promotes recovery and limits neurodegeneration, but this is inefficient and always ultimately fails. Furthermore, these regenerated myelin sheaths are thinner and shorter than the original, leaving the underlying axons potentially vulnerable. In rodent models, CNS remyelination is more efficient, so that in young animals (but not old) the number of myelinated axons is efficiently restored to normal, but in both young and old rodents, regenerated myelin sheaths are still short and thin. The reasons for these differences in remyelination efficiency, the thinner remyelinated myelin sheaths compared to developmental myelin and the subsequent effect on the underlying axon are unclear. We studied CNS remyelination in the highly regenerative adult zebrafish (Danio rerio), to better understand mechanisms of what we hypothesised would be highly efficient remyelination, and to identify differences to mammalian CNS remyelination, as larval zebrafish are increasingly used for high throughput screens to identify potential drug targets to improve myelination and remyelination. RESULTS: We developed a novel method to induce a focal demyelinating lesion in adult zebrafish optic nerve with no discernible axonal damage, and describe the cellular changes over time. Remyelination is indeed efficient in both young and old adult zebrafish optic nerves, and at 4 weeks after demyelination, the number of myelinated axons is restored to normal, but internode lengths are short. However, unlike in rodents or in humans, in young zebrafish these regenerated myelin sheaths were of normal thickness, whereas in aged zebrafish, they were thin, and remained so even 3 months later. This inability to restore normal myelin thickness in remyelination with age was associated with a reduced macrophage/microglial response. CONCLUSION: Zebrafish are able to efficiently restore normal thickness myelin around optic nerve axons after demyelination, unlike in mammals. However, this fails with age, when only thin myelin is achieved. This gives us a novel model to try and dissect the mechanism for restoring myelin thickness in CNS remyelination. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40478-014-0077-y) contains supplementary material, which is available to authorized users

Anatomy and pharmacology of vessels

A typical blood vessel is arranged in three distinct layers forming concentric circles. The innermost layer in contact with the blood is composed of endothelial cells on a basement membrane through which the endothelial cells and underlying vascular smooth muscle cells of the medial layer can communicate. The endothelium releases mediators and factors that have effects on vessel diameter, platelet adhesion and thrombus formation and inflammatory cell adhesion. The thickness and elasticity of the medial smooth muscle layer is dependent on the position of the vessel within the vascular tree and is adapted to the function of the vessel. The adventitia contains neural varicosities that modulate vessel diameter, and recent work has highlighted a paracrine role of the adipose tissue surrounding blood vessels. The adventitia also contains fibroblasts and inflammatory cells, such as resident macrophages. This chapter discusses blood vessel structure and function with a particular focus on how physiological mediators alter vessel diameter and how this function can go awry in some common cardiovascular diseases