Plxdc2 is a mitogen for neural progenitors

The development of different brain regions involves the coordinated control of proliferation and cell fate specification along and across the neuraxis. Here, we identify Plxdc2 as a novel regulator of these processes, using in ovo electroporation and in vitro cultures of mammalian cells. Plxdc2 is a type I transmembrane protein with some homology to nidogen and to plexins. It is expressed in a highly discrete and dynamic pattern in the developing nervous system, with prominent expression in various patterning centres. In the chick neural tube, where Plxdc2 expression parallels that seen in the mouse, misexpression of Plxdc2 increases proliferation and alters patterns of neurogenesis, resulting in neural tube thickening at early stages. Expression of the Plxdc2 extracellular domain alone, which can be cleaved and shed in vivo, is sufficient for this activity, demonstrating a cell non-autonomous function. Induction of proliferation is also observed in cultured embryonic neuroepithelial cells (ENCs) derived from E9.5 mouse neural tube, which express a Plxdc2-binding activity. These experiments uncover a direct molecular activity of Plxdc2 in the control of proliferation, of relevance in understanding the role of this protein in various cancers, where its expression has been shown to be altered. They also implicate Plxdc2 as a novel component of the network of signalling molecules known to coordinate proliferation and differentiation in the developing nervous system.     

Expression of the LIM-homeodomain gene Lmx1a (dreher) during development of the mouse nervous system

The expression pattern of Lmx1a, a LIM-homeodomain gene disrupted in the dreher mouse neurological mutant, is described during development. Lmx1a is predominantly expressed in the developing nervous system from embryonic day E8.5 to adulthood, in restricted areas. Major expression domains include the dorsal midline (roof plate) of the neural tube, the cortical hem, the otic vesicles, the developing cerebellum and the notochord. The Lmx1a expression pattern is therefore well correlated with the various aspects of the phenotype of the dreher mutant mice.     

Wnt signaling specifies and patterns intestinal endoderm

Wnt signaling has been implicated in many developmental processes, but its role in early endoderm development is not well understood. Wnt signaling is active in posterior endoderm as early as E7.5. Genetic and chemical activation show that the Wnt pathway acts directly on endoderm to induce the intestinal master regulator Cdx2, shifting global gene away from anterior endoderm and toward a posterior, intestinal program. In a mouse embryonic stem cell differentiation platform that yields pure populations of definitive endoderm, Wnt signaling induces intestinal gene expression in all cells. We have identified a set of genes specific to the anterior small intestine, posterior small intestine, and large intestine during early development, and show that Wnt, through Cdx2, activates large intestinal gene expression at high doses and small intestinal gene expression at lower doses. These findings shed light on the mechanism of embryonic intestinal induction and provide a method to manipulate intestinal development from embryonic stem cells.     

Development of the mammalian cortical hem and its derivatives: the choroid plexus,Cajal–Retzius cells and hippocampus

The dorsal medial region of the developing mammalian telencephalon plays a central role in the patterning of the adjacent brain regions. This review describes the development of this specialized region of the vertebrate brain, called the cortical hem, and the formation of the various cells and structures it gives rise to, including the choroid plexus, Cajal–Retzius cells and the hippocampus. We highlight the ontogenic processes that create these different forebrain derivatives from their shared embryonic origin and discuss the key signalling pathways and molecules that influence the patterning of the cortical hem. These include BMP, Wnt, FGF and Shh signalling pathways acting with Homeobox factors to carve the medial telencephalon into district progenitor regions, which in turn give rise to the choroid plexus, dentate gyrus and hippocampus. We then link the formation of the lateral ventricle choroid plexus with embryonic and postnatal neurogenesis in the hippocampus.     

The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells

Neural precursor cells (NPCs) have the ability to self-renew and to give rise to neuronal and glial lineages. The fate decision of NPCs between proliferation and differentiation determines the number of differentiated cells and the size of each region of the brain. However, the signals that regulate the timing of neuronal differentiation remain unclear. Here, we show that Wnt signaling inhibits the self-renewal capacity of mouse cortical NPCs, and instructively promotes their neuronal differentiation. Overexpression of Wnt7a or of a stabilized form of beta-catenin in mouse cortical NPC cultures induced neuronal differentiation even in the presence of Fgf2, a self-renewal-promoting factor in this system. Moreover, blockade of Wnt signaling led to inhibition of neuronal differentiation of cortical NPCs in vitro and in the developing mouse neocortex. Furthermore, the beta-catenin/TCF complex appears to directly regulate the promoter of neurogenin 1, a gene implicated in cortical neuronal differentiation. Importantly, stabilized beta-catenin did not induce neuronal differentiation of cortical NPCs at earlier developmental stages, consistent with previous reports indicating self-renewal-promoting functions of Wnts in early NPCs. These findings may reveal broader and stage-specific physiological roles of Wnt signaling during neural development.     

Duplicate sfrp1 genes in zebrafish: sfrp1a is dynamically expressed in the developing central nervous system, gut and lateral line

The secreted frizzled-related proteins (Sfrp) are a family of soluble proteins with diverse biological functions having the capacity to bind Wnt ligands, to modulate Wnt signalling, and to signal directly via the Wnt receptor, Frizzled. In an enhancer trap screen for embryonic expression in zebrafish we identified an sfrp1 gene. Previous studies suggest an important role for sfrp1 in eye development, however, no data have been reported using the zebrafish model. In this paper, we describe duplicate sfrp1 genes in zebrafish and present a detailed analysis of the expression profile of both genes. Whole mount in situ hybridisation analyses of sfrp1a during embryonic and larval development revealed a dynamic expression profile, including: the central nervous system, where sfrp1a was regionally expressed throughout the brain and developing eye; the posterior gut, from the time of endodermal cell condensation; the lateral line, where sfrp1a was expressed in the migrating primordia and interneuromast cells that give rise to the sensory organs. Other sites included the blastoderm, segmenting mesoderm, olfactory placode, developing ear, pronephros and fin-bud. We have also analysed sfrp1b expression during embryonic development. Surprisingly this gene exhibited a divergent expression profile being limited to the yolk syncytium under the elongating tail-bud, which later covered the distal yolk extension, and transiently in the tail-bud mesenchyme. Overall, our studies provide a basis for future analyses of these developmentally important factors using the zebrafish model.     

Interfering with Wnt signalling alters the periodicity of the segmentation clock

Somites are embryonic precursors of the ribs, vertebrae and certain dermis tissue. Somite formation is a periodic process regulated by a molecular clock which drives cyclic expression of a number of clock genes in the presomitic mesoderm. To date the mechanism regulating the period of clock gene oscillations is unknown. Here we show that chick homologues of the Wnt pathway genes that oscillate in mouse do not cycle across the chick presomitic mesoderm. Strikingly we find that modifying Wnt signalling changes the period of Notch driven oscillations in both mouse and chick but these oscillations continue. We propose that the Wnt pathway is a conserved mechanism that is involved in regulating the period of cyclic gene oscillations in the presomitic mesoderm.     

Wnt signaling is required at distinct stages of development for the induction of the posterior forebrain

One of the earliest manifestations of anteroposterior pattering in the developing brain is the restricted expression of Six3 and Irx3 in the anterior and posterior forebrain, respectively. Consistent with the role of Wnts as posteriorizing agents in neural tissue, we found that Wnt signaling was sufficient to induce Irx3 and repress Six3 expression in forebrain explants. The position of the zona limitans intrathalamica (zli), a boundary-cell population that develops between the ventral (vT) and dorsal thalamus (dT), is predicted by the apposition of Six3 and Irx3 expression domains. The expression patterns of several inductive molecules are limited by the zli, including Wnt3, which is expressed posterior to the zli in the dT. Wnt3 and Wnt3a were sufficient to induce the dT marker Gbx2 exclusively in explants isolated posterior to the presumptive zli. Blocking the Wnt response allowed the induction of the vT-specific marker Dlx2 in prospective dT tissue. Misexpression of Six3 in the dT induced Dlx2 expression and inhibited the expression of both Gbx2 and Wnt3. These results demonstrate a dual role for Wnt signaling in forebrain development. First, Wnts directed the initial expression of Irx3 and repression of Six3 in the forebrain, delineating posterior and anterior forebrain domains. Later, continued Wnt signaling resulted in the induction of dT specific markers, but only in tissues that expressed Irx3.     

Genetic damage and repair in human rectal cells for biomonitoring: sex differences, effects of alcohol exposure, and susceptibilities in comparison to peripheral blood lymphocytes

Harlequin (Hq) mice develop ataxia due to an X-linked recessive mutation in the gene encoding apoptosis-inducing factor (Aif). Brain cells in Hq mice contain the modified base 8-hydroxydeoxyguanosine (8-OHdG), suggesting that the defect in Aif causes increased DNA oxidation in these cells. Because oxidative damage is mutagenic, Hq mice might suffer increased mutation in the brain. To examine this possibility, mutation in the brain was assessed using the Tg(βA-G11PLAP) mouse model, which allows mutant cells to be visualized in tissue sections in situ. Hq mice exhibited more and larger patches of PLAP positive tissue in the brain. PLAP+ cells were observed in all areas of the brain. No increase in the number of PLAP+ cells was seen in three other tissues, suggesting that the effect of Aif deficiency on mutation was specific to brain.     

Fgf8 controls regional identity in the developing thalamus

The vertebrate thalamus contains multiple sensory nuclei and serves as a relay station to receive sensory information and project to corresponding cortical areas. During development, the progenitor region of the diencephalon is divided into three parts, p1, p2 (presumptive thalamus) and p3, along its longitudinal axis. Besides the local expression of signaling molecules such as sonic hedgehog (Shh), Wnt proteins and Fgf8, the patterning mechanisms of the thalamic nuclei are largely unknown. Using mouse in utero electroporation to overexpress or inhibit endogenous Fgf8 at the diencephalic p2/p3 border, we revealed that it affected gene expression only in the p2 region without altering overall diencephalic size or the expression of other signaling molecules. We demonstrated that two distinctive populations in p2, which can be distinguished by Ngn2 and Mash1 in early embryonic diencephalon, are controlled by Fgf8 activity in complementary manner. Furthermore, we found that FGF activity shifts thalamic sensory nuclei on the A/P axis in postnatal brain. Moreover, gene expression analysis demonstrated that FGF signaling shifts prethalamic nuclei in complementary manner to the thalamic shift. These findings suggest conserved roles of FGF signaling in patterning along the A/P axis in CNS, and reveal mechanisms of nucleogenesis in the developing thalamus.     

Colocalization and redistribution of dishevelled and actin during Wnt-induced mesenchymal morphogenesis

Activation of the Wnt signaling pathway is important for induction of gene expression and cell morphogenesis throughout embryonic development. We examined the subcellular localization of dishevelled, the immediate downstream component from the Wnt receptor, in the embryonic mouse kidney. Using immunofluorescence staining, confocal microscopy, and coimmunoprecipitation experiments, we show that dishevelled associates with actin fibers and focal adhesion plaques in metanephric mesenchymal cells. Stimulation of Wnt signaling leads to profound changes in metanephric mesenchymal cell morphology, including disruption of the actin cytoskeleton, increased cell spreading, and increased karyokinesis. Upon activation of Wnt signaling, dishevelled also accumulates in and around the nucleus. Casein kinase Iepsilon colocalizes with dishevelled along actin fibers and in the perinuclear region, whereas axin and GSK-3 are only present around the nucleus. These data indicate a branched Wnt signaling pathway comprising a canonical signal that targets the nucleus and gene expression, and another signal that targets the cytoskeleton and regulates cell morphogenesis.     

All four zebrafish Wnt7 genes are expressed during early brain development

Wnt-signalling is involved in a number of biological processes in the course of embryonic development, cell fate determination, proliferation, stem cell maintenance and oncogenesis. Wnt ligands are secreted glycoproteins and the number of Wnt isoforms varies between five in nematodes and 27 in fish. The highly conserved group of Wnt7 genes has been found to signal via at least three Wnt-signalling pathways dependent on the developmental context. These ligands have been identified as important regulators in a number of processes ranging from formation of bones, lungs, kidneys, reproductive organs and placenta to vasculogenesis and synaptogenesis in the brain. The importance of Wnt7 function is underscored by their implication in disease syndromes in man. Unlike the single Wnt7a and Wnt7b mammalian genes we find that the zebrafish genome contains two paralogues genes for each Wnt7 ligand. Here, we compare these four Wnt7 genes evolutionarily and analyse their expression during the first two days of embryonic development. We find Wnt7 genes mainly expressed in a number of CNS structures at developmental stages at which patterning and neural specification takes place. The timely and spatially overlapping as well as complementary gene expression suggests diverse as well as redundant involvements during brain development.