Michael Lardelli

Nestin mRNA expression correlates with the central nervous system progenitor cell state in many, but not all, regions of developing central nervous system

Nestin is a recently discovered intermediate filament (IF) gene. Nestin expression has been extensively used as a marker for central nervous system (CNS) progenitor cells in different contexts, based on observations indicating a correlation between nestin expression and this cell type in vivo. To evaluate this correlation in more detail nestin mRNA expression in developing and adult mouse CNS was analysed by in situ hybridization. We find that nestin is expressed from embryonic day (E) 7.75 and that expression is detected in many proliferating CNS regions: at E10.5 nestin is expressed in cells of both the rostral and caudal neural tube, including the radial glial cells; at E15.5 and postnatal day (P) 0 expression is observed largely in the developing cerebellum and in the ventricular and subventricular areas of the developing telencephalon. Furthermore, the transition from a proliferating to a post-mitotic cell state is accompanied by a rapid decrease in nestin mRNA for motor neurons in the ventral spinal cord and for neurons in the marginal layer of developing telencephalon. In contrast to these data we observe two proliferating areas, the olfactory epithelium and the precursor cells of the hippocampal granule neurons, which do not express nestin at detectable levels. Thus, nestin mRNA expression correlates with many, but not all, regions of proliferating CNS progenitor cells. In addition to its temporal and spatial regulation nestin expression also appears to be regulated at the level of subcellular mRNA localization: in columnar neuroepithelial and radial glial cells nestin mRNA is predominantly localized to the pial endfeet.

The novel Notch homologue mouse Notch 3 lacks specific epidermal growth factor-repeats and is expressed in proliferating neuroepithelium

In Drosophila, the Notch gene is pivotal for cell fate decisions at many stages of development and, in particular, during the formation of the nervous system. Absence of Notch results in the generation of excessive numbers of neural cells at the expense of epidermal cells. Two previously identified mammalian Notch homologous encode all the principal features of the Drosophila gene, e.g. 36 EGF-repeats and 3 Notch/lin-12 repeats extracellularly and 6 intracellular cdc10/SWI6 repeats. We report here the characterisation of a third mammalian homologue, mouse Notch 3, which shares the same remarkable conservation relative to the Drosophila gene as the two previously identified homologues, but with three important distinctions. First, Notch 3 specifically lacks the equivalent of EGF-repeat 21; second, it lacks an EGF-repeat-sized region comprising parts of EGF-repeats 2 and 3; and third, it encodes a considerably shorter intracellular domain. The Notch 3 gene is expressed at high levels in proliferating neuroepithelium and expression is downregulated at later stages. The expression patterns of the Notch 1, 2 and 3 genes are quite distinct during central nervous system (CNS) development, and all possible combinations of expression, i.e. none, one, two, or all three genes, are seen, suggesting a combinatorial code of Notch function in mammals. Considering the predominantly early expression in CNS and its distinct structural features, the Notch 3 gene is likely to contribute significantly to vertebrate Notch function during CNS development.

The Drosophila hairy protein acts in both segmentation and bristle patterning and shows homology to N-myc.

The Drosophila segmentation gene, hairy (h), acts to regulate embryonic segmentation and bristle pattern. We present the DNA sequence of the h gene and of h cDNAs, thereby deducing the organization of the h transcripts. The h gene encodes a 337 amino acid protein that acts in both embryonic segmentation and adult bristle patterning. The h protein includes a domain that shows extensive similarity to a domain of the proto‐oncogene N‐myc that may be involved in DNA binding and/or protein dimerization. We discuss mechanisms of h action as a transcriptional regulator.

Complementary and combinatorial patterns of Notch gene family expression during early mouse development

The Drosophila Notch gene encodes a transmembrane receptor involved in the regulation of cell fate. It exerts its effect by lateral specification, inductive signaling and is also important for cell adhesion and axonal pathfinding. In this report we analyse the expression of the three mammalian Notch homologues during early mouse development by in situ hybridization. The Notch 1, 2 and 3 genes show dynamic and complex expression patterns, in particular during gastrulation and somitogenesis and in early nervous system formation. During gastrulation, the Notch genes are expressed in non-overlapping, successive patterns. Notch 3 is widely expressed in both ectoderm and mesoderm. Notch 2 is then expressed in the node, notochord and neural groove while Notch 1 becomes highly expressed in presomitic mesoderm. As somitogenesis begins, Notch 2 expression is activated in newly forming somites while Notch 3 is activated in mature somites. Various neural crest cell populations and ectodermal placode cells can be defined by expression of specific combinations of Notch genes. All three Notch genes are expressed within cells of the dorsal neural tube at E9.5, although neural crest cells that have begun migrating all show distinct patterns of Notch expression. Finally, Notch 1 expression is observed not only in placodes, but also in cells migrating from placodes to the site of the ganglia anlagen. This expression pattern may be analogous to Notch expression in the peripheral nervous system of Drosophila, suggesting that mammalian Notch genes may also be involved in axonal pathfinding.

The Guinea Pig as a Model for Sporadic Alzheimer's Disease (AD): The Impact of Cholesterol Intake on Expression of AD-Related Genes

We investigated the guinea pig, Cavia porcellus, as a model for Alzheimer’s disease (AD), both in terms of the conservation of genes involved in AD and the regulatory responses of these to a known AD risk factor - high cholesterol intake. Unlike rats and mice, guinea pigs possess an Aβ peptide sequence identical to human Aβ. Consistent with the commonality between cardiovascular and AD risk factors in humans, we saw that a high cholesterol diet leads to up-regulation of BACE1 (β-secretase) transcription and down-regulation of ADAM10 (α-secretase) transcription which should increase release of Aβ from APP. Significantly, guinea pigs possess isoforms of AD-related genes found in humans but not present in mice or rats. For example, we discovered that the truncated PS2V isoform of human PSEN2, that is found at raised levels in AD brains and that increases γ-secretase activity and Aβ synthesis, is not uniquely human or aberrant as previously believed. We show that PS2V formation is up-regulated by hypoxia and a high-cholesterol diet while, consistent with observations in humans, Aβ concentrations are raised in some brain regions but not others. Also like humans, but unlike mice, the guinea pig gene encoding tau, MAPT, encodes isoforms with both three and four microtubule binding domains, and cholesterol alters the ratio of these isoforms. We conclude that AD-related genes are highly conserved and more similar to human than the rat or mouse. Guinea pigs represent a superior rodent model for analysis of the impact of dietary factors such as cholesterol on the regulation of AD-related genes.

Three novel Notch genes in zebrafish: implications for vertebrate Notch gene evolution and function

Abstract Notch genes encode transmembrane receptors that interact with numerous signal transduction pathways and are essential for animal development. To facilitate analysis of vertebrate Notch gene function, we isolated cDNA fragments of three novel Notch genes from zebrafish (Danio rerio), Notch1b, Notch5 and Notch6. Notch1b is a second zebrafish Notch1 gene. From analysis of the Notch1b sequence we argue that the various vertebrate Notch gene subfamilies encode receptors with different signalling specificities. Notch5 and Notch6 represent novel vertebrate Notch gene subfamilies. Remarkably, Notch1b lacks expression in presomitic mesoderm, Notch5 is expressed in a metameric pattern within the presomitic mesoderm whilst Notch6 expression is excluded from the nervous system. The expression patterns of these genes suggest important roles in gastrulation, somitogenesis, tail bud extension, myogenesis, heart development and neurogenesis. We discuss the implications of our observations for Notch gene evolution and function.

Expression of the Notch 3 intracellular domain in mouse central nervous system progenitor cells is lethal and leads to disturbed neural tube development

Notch-like receptors are found in organisms ranging from nematodes to mammals. In Drosophila, Notch plays a key role in cell fate decisions in the early nervous system. In this report we analyse the effects of excess Notch 3 activity in central nervous system (CNS) progenitor cells. A mutated Notch gene encoding the intracellular domain of mouse Notch 3 transcribed from the nestin promoter was expressed in CNS progenitor cells in transgenic mice. This mutation resulted in a phenotypic series of neural tube defects in embryonic day 10.5-12.5 embryos and proved lethal to embryos beyond this age. In the milder phenotype the neural tube displayed a zig-zag morphology and the CNS was slightly enlarged. More severely affected embryos showed a lack of closure of the anterior neural pore, resulting in the externalization of neural tissue and the complete collapse of the third and fourth ventricles. The expanded ventricular zone of the neuroepithelium, a correspondingly enlarged area of nestin expression, and an increase in the number of proliferating cells in the neural tube suggested that these phenotypes resulted from an expanded CNS progenitor cell population. These data provide support in vivo for the notion that Notch activity plays a role in mammalian CNS development and may be required to guide CNS progenitor cells in their choice between continued proliferation or neuronal differentiation.

Thyroid abnormalities and hepatocellular carcinoma in mice transgenic for v-erbA.

The v‐erbA oncogene consists of an avian retroviral gag gene fused to a mutated thyroid hormone receptor. To define better its role as an oncogene in mammals and its ability to function as a dominant negative transcription factor, transgenic mice expressing v‐erbA ubiquitously were generated. The effects of v‐erbA are pleiotropic, tissue‐specific and dose dependent. Mice have breeding disorders, abnormal behavior, reduced adipose tissue, hypothyroidism with inappropriate TSH response, and enlarged seminal vesicles. This provides an animal model consistent with the proposal that v‐ErbA functions as a dominant negative receptor by transcriptional interference or squelching of normal receptors or associated proteins. Finally, male animals develop hepatocellular carcinoma, demonstrating that v‐erbA can promote neoplasia in mammals.

The protein tyrosine phosphatase Pez regulates TGFβ, epithelial–mesenchymal transition, and organ development

Epithelial–mesenchymal transition (EMT), crucial during embryogenesis for new tissue and organ formation, is also considered to be a prerequisite to cancer metastasis. We report here that the protein tyrosine phosphatase Pez is expressed transiently in discrete locations in developing brain, heart, pharyngeal arches, and somites in zebrafish embryos. We also find that Pez knock-down results in defects in these organs, indicating a crucial role in organogenesis. Overexpression of Pez in epithelial MDCK cells causes EMT, with a drastic change in cell morphology and function that is accompanied by changes in gene expression typical of EMT. Transfection of Pez induced TGFβ signaling, critical in developmental EMT with a likely role also in oncogenic EMT. In zebrafish, TGFβ3 is co- expressed with Pez in a number of tissues and its expression was lost from these tissues when Pez expression was knocked down. Together, our data suggest Pez plays a crucial role in organogenesis by inducing TGFβ and EMT.

Zebrafish fgfr1 is a member of the fgf8 synexpression group and is required for fgf8 signalling at the midbrain-hindbrain boundary

FGFR1 is an important signalling molecule during embryogenesis and in adulthood. FGFR1 mutations in human may lead to developmental defects and pathological conditions, including cancer and Alzheimer’s disease. Here, we describe cloning and expression analysis of the zebrafish fibroblast growth factor receptor 1 (fgfr1). Initially, fgfr1 is expressed in the adaxial mesoderm with transcripts distinctly localised to the anterior portion of each half-somite. Hereupon, fgfr1 is also strongly expressed in the otic vesicles, branchial arches and the brain, especially at the midbrain-hindbrain boundary (MHB). The expression patterns of fgfr1 and fgf8 are strikingly similar and knock-down of fgfr1 phenocopies many aspects observed in the fgf8 mutant acerebellar, suggesting that Fgf8 exerts its function mainly by binding to FgfR1.