A transgenic Cre mouse line for the study of cortical and hippocampal development

Wnt signaling regulates cortical and hippocampal development. In a previous study we found that a particular Wnt receptor, Frizzled9 (Fzd9), was selectively expressed in both the developing and adult hippocampus. Taking advantage of the specificity of this promoter, we generated a transgenic cre mouse line using the putative control elements of the Fzd9 gene. In the Fzd9‐cre mice, Cre is mainly detected in the developing cortex and hippocampus and is confined to the CA fields and dentate gyrus in adults. Furthermore, by crossing the Fzd9‐cre mouse with the ROSA26 reporter line, we examined the activity of Cre and found that it has very high recombination efficiency. Thus, this mouse line will likely prove to be a useful tool for studying cortical and hippocampal development via activation or inactivation of interesting genes. genesis 48:343–350, 2010. © 2010 Wiley‐Liss, Inc.     

Wnt7a Regulates Multiple Steps of Neurogenesis

Although Wnt7a has been implicated in axon guidance and synapse formation, investigations of its role in the early steps of neurogenesis have just begun. We show here that Wnt7a is essential for neural stem cell self-renewal and neural progenitor cell cycle progression in adult mouse brains. Loss of Wnt7a expression dramatically reduced the neural stem cell population and increased the rate of cell cycle exit in neural progenitors in the hippocampal dentate gyrus of adult mice. Furthermore, Wnt7a is important for neuronal differentiation and maturation. Loss of Wnt7a expression led to a substantial decrease in the number of newborn neurons in the hippocampal dentate gyrus. Wnt7a^−/− dentate granule neurons exhibited dramatically impaired dendritic development. Moreover, Wnt7a activated β-catenin and its downstream target genes to regulate neural stem cell proliferation and differentiation. Wnt7a stimulated neural stem cell proliferation by activating the β-catenin–cyclin D1 pathway and promoted neuronal differentiation and maturation by inducing the β-catenin–neurogenin 2 pathway. Thus, Wnt7a exercised critical control over multiple steps of neurogenesis by regulating genes involved in both cell cycle control and neuronal differentiation.     

Alterations of Glial Cells in the Mouse Hippocampus During Postnatal Development

We investigated the postnatal alterations of neurons, astrocyte, oligodendrocyte, and microglia in the mouse hippocampal CA1 sector and dentate gyrus under the same conditions using immunohistochemistry. Neuronal nuclei (NeuN), Glial fibrillary acidic protein (GFAP), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), and ionized calcium binding adaptor molecule 1 (Iba 1) immunoreactivity were measured in 1-, 2-, 4-, and 8-week-old mice. Total number of NeuN-positive neurons was unchanged in the mouse hippocampal CA1 sector and dentate gyrus from 1 to 8 weeks of birth. In contrast, a significant increase in the number of GFAP-positive astrocytes was observed only in the hippocampal CA1 sector of 1-week-old mice when compared with 8-week-old animals. Thereafter, total number of GFAP-positive astrocytes was unchanged in the hippocampal CA1 sector and dentate gyrus from 2 to 8 weeks of birth. For microglia, a significant increase in the number of Iba 1-positive microglia was observed in the hippocampal CA1 sector and dentate gyrus of 1-, 2-, and 4-week-old mice as compared with 8-week-old animals. On the other hand, a significant decrease in the area of expression of CNPase-positive fibers was observed in the hippocampal CA1 sector of 1- and 2-week-old mice as compared with 8-week-old animals. In dentate gyrus, a significant decrease in the area of expression of CNPase-positive fibers was found in 1-, 2-, and 4-week-old mice. Furthermore, our double-labeled immunostaining showed that brain-derived neurotrophic factor (BDNF) immunoreactivity was observed in GFAP-positive astrocytes and Iba 1-positive microglia in the hippocampal CA1 sector and dentate gyrus of 1- and 2-week-old mice. These results show that glial cells may play some role in the maintenance and neuronal functions of hippocampal CA1 pyramidal neurons and granule cells of dentate gyrus during postnatal development. Furthermore, our results demonstrate that glial BDNF may play an important role in the maturation of oligodendrocyte in the hippocampal CA1 sector and dentate gyrus during postnatal development. Thus, our findings provide valuable information on the developmental processes.     

An inducible transgenic Cre mouse line for the study of hippocampal development and adult neurogenesis

The hippocampus is crucial for higher brain functions, such as learning, memory, and emotion. Many diseases like epilepsy and Down's syndrome are associated with abnormalities in early hippocampal development. In addition, adult dentate neurogenesis is thought to be defective in several classes of psychiatric disorders. However, the mechanisms regulating hippocampal development and adult neurogenesis remain unclear. One of the limitations to studying these processes is the scarcity of available specific mouse tools. Here, we report an inducible transgenic Cre mouse line, Frizzled 9‐CreER?, in which tamoxifen administration induces Cre recombinant. Our data show that Cre is expressed in the developing hippocampal primordium, confined to the granule cell layer at P20 and further limited to the subgranular zone in the adult dentate gyrus. Cre recombinase shows very high activity in all of these regions. Thus, this transgenic line will be a powerful tool in understanding the mechanisms of hippocampal development, adult neurogenesis, and associated diseases. genesis 49:919–926, 2011. © 2011 Wiley Periodicals, Inc.     

Long-Term Fate Mapping Using Conditional Lentiviral Vectors Reveals a Continuous Contribution of Radial Glia-Like Cells to Adult Hippocampal Neurogenesis in Mice

Newborn neurons are generated throughout life in two neurogenic regions, the subventricular zone and the hippocampal dentate gyrus. Stimulation of adult neurogenesis is considered as an attractive endogenous repair mechanism to treat different neurological disorders. Although tremendous progress has been made in our understanding of adult hippocampal neurogenesis, important questions remain unanswered, regarding the identity and the behavior of neural stem cells in the dentate gyrus. We previously showed that conditional Cre-Flex lentiviral vectors can be used to label neural stem cells in the subventricular zone and to track the migration of their progeny with non-invasive bioluminescence imaging. Here, we applied these Cre-Flex lentiviral vectors to study neurogenesis in the dentate gyrus with bioluminescence imaging and histological techniques. Stereotactic injection of the Cre-Flex vectors into the dentate gyrus of transgenic Nestin-Cre mice resulted in specific labeling of the nestin-positive neural stem cells. The labeled cell population could be detected with bioluminescence imaging until 9 months post injection, but no significant increase in the number of labeled cells over time was observed with this imaging technique. Nevertheless, the specific labeling of the nestin-positive neural stem cells, combined with histological analysis at different time points, allowed detailed analysis of their neurogenic potential. This long-term fate mapping revealed that a stable pool of labeled nestin-positive neural stem cells continuously contributes to the generation of newborn neurons in the mouse brain until 9 months post injection. In conclusion, the Cre-Flex technology is a valuable tool to address remaining questions regarding neural stem cell identity and behavior in the dentate gyrus.     

Spatiotemporal expression pattern of Sjfz7 and its expression comparison with other frizzled family genes in developmental stages of Schistosoma japonicum

Wnts are secreted signaling molecules that are implicated in a variety of growth-related processes. Frizzled proteins have been identified as receptors for Wnt ligands in vertebrates and invertebrates, but a functional role for dioecious flatworm Frizzleds has not been determined. To evaluate the endogenous role of Frizzled proteins during development, we have identified and characterized a Schistosoma japonicum frizzled gene (Sjfz7). We found that Sjfz7 encodes a 698 amino acid protein with typical characteristics of Frizzled proteins. The immunohistochemical localization pattern showed that Sjfz7 protein was extensively distributed in almost all tissues of S. japonicum, including subtegumental muscle cells, parenchymal cells, intestinal epithelial cells and male and female germ cells. This indicated that Sjfz7-mediated Wnt signaling might be associated with the development of musculature, intestinal tract and reproductive organs in schistosome. Comparing mRNA levels between frizzled family members showed that Sjfz7 mRNA was consistently higher in the developmental stages analyzed, suggesting that Sjfz7 may be responsible for more functional tasks than other frizzled family members. Comparing frizzled mRNA levels between not fully developed and normal worms suggested that Wnt signaling might be abnormal in not fully developed worms.     

Noggin and BMP4 co-modulate adult hippocampal neurogenesis in the APPswe/PS1ΔE9 transgenic mouse model of Alzheimer’s disease

In addition to the subventricular zone, the dentate gyrus of the hippocampus is one of the few brain regions in which neurogenesis continues into adulthood. Perturbation of neurogenesis can alter hippocampal function, and previous studies have shown that neurogenesis is dysregulated in Alzheimer disease (AD) brain. Bone morphogenetic protein-4 (BMP4) and its antagonist Noggin have been shown to play important roles both in embryonic development and in the adult nervous system, and may regulate hippocampal neurogenesis. Previous data indicated that increased expression of BMP4 mRNA within the dentate gyrus might contribute to decreased hippocampal cell proliferation in the APP_swe/PS1_ΔE9 mouse AD model. However, it is not known whether the BMP antagonist Noggin contributes to the regulation of neurogenesis. We therefore studied the relative expression levels and localization of BMP4 and its antagonist Noggin in the dentate gyrus and whether these correlated with changes in neurogenesis in 6-12 mo old APP_swe/PS1_ΔE9 transgenic mice. Bromodeoxyuridine (BrdU) was used to label proliferative cells. We report that decreased neurogenesis in the APP/PS1 transgenic mice was accompanied by increased expression of BMP4 and decreased expression of Noggin at both the mRNA and protein levels; statistical analysis showed that the number of proliferative cells at different ages correlated positively with Noggin expression and negatively with BMP4 expression. Intraventricular administration of a chimeric Noggin/Fc protein was used to block the action of endogenous BMP4; this resulted in a significant increase in the number of BrdU-labeled cells in dentate gyrus subgranular zone and hilus in APP/PS1 mice. These results suggest that BMP4 and Noggin co-modulate neurogenesis.     

sFRP3 inhibition improves age‐related cellular changes in BubR1 progeroid mice

Wnt signaling is a well‐known molecular pathway in age‐related pathogenesis and therapy of disease. While prior studies have mainly focused on Wnt ligands or Wnt activators, the in vivo functions of naturally secreted Wnt inhibitors are not clear, especially in brain aging. Using BubR1^H/H mice as a novel mouse model of accelerated aging, we report that genetic inhibition of sFRP3 restores the reduced body and brain size observed in BubR1^H/H mice. Furthermore, sFRP3 inhibition ameliorates hypomyelination in the corpus callosum and rescues neural progenitor proliferation in the hippocampal dentate gyrus of BubR1^H/H mice. Taken together, our study identifies sFRP3 as a new molecular factor that cooperates with BubR1 function to regulate brain development, myelination, and hippocampal neurogenesis.     

Xenopus frizzled-2 is expressed highly in the developing eye, otic vesicle and somites.

Wnts are secreted signaling molecules implicated in a large number of developmental processes. Frizzled proteins have been identified as likely receptors for Wnt ligands in vertebrates and invertebrates. To assess the endogenous role of frizzled proteins during the development of Xenopus laevis, we have identified several frizzled homologs. Here we report the cloning and expression of Xenopus frizzled-2 (xfz2). Xfz2 shows high sequence homology to rat and human frizzleds-2. It is expressed in the developing embryo from late gastrula stages onward. Xfz2 has a wide domain of expression but is concentrated in the eye anlage, otic vesicle, and developing somites.     

Ligand receptor interactions in the Wnt signaling pathway in Drosophila

Secreted Wnt proteins have numerous signaling functions during development, mediated by Frizzled molecules that act as Wnt receptors on the cell surface. In the genome of Drosophila, seven Wnt genes (including wingless; wg), and five frizzled genes have been identified. Relatively little is known about signaling and binding specificities of different Wnt and Frizzled proteins. We have developed an assay to determine the strength of binding between membrane-tethered Wnts and ligand binding domains of the Frizzled receptors. We found a wide spectrum of binding affinities, reflecting known genetic interactions. Most Wnt proteins can bind to multiple Frizzleds and vice versa, suggesting redundancy in vivo. In an extension of these experiments, we tested whether two different subdomains of the Wg protein would by themselves bind to Frizzled and generate a biological response. Whereas these two separate domains are secreted from cells, suggesting that they form independently folded parts of the protein, they were only able to evoke a response when co-transfected, indicating that both are required for function. In addition to the Frizzleds, members of the LRP family (represented by the arrow gene in Drosophila) are also necessary for Wnt signal transduction and have been postulated to act as co-receptors. We have therefore examined whether a soluble form of the Arrow molecule can bind to Wingless and Frizzled, but no interactions were detected.     

Frizzled-9 promoter drives expression of transgenes in the medial wall of the cortex and its chief derivative the hippocampus

The hippocampus develops from the medial wall of the forming cerebral cortex during embryonic life. Morphogenic signals from the Wnt pathway regulate several events during hippocampal development (Galceran et al.: Development 127:469-482, 2000; Lee et al.: Development 127:457-467, 2000; Zhou et al.: J Neurosci 24:121-126, 2004) and we have previously shown that Wnt receptors from the Frizzled (Fzd) family are expressed in discreet cortical domains during development (Kim et al.: Mech Dev 103:167-172, 2001). We generated transgenic mice using the putative control elements of the Fzd9 gene, normally selectively expressed in the developing and adult hippocampus, driving expression of a marker gene. These mice express LacZ in the brain in the same developmental distribution as endogenous Fzd protein. Postnatally, expression remains strong in the dendritic fields of hippocampal principal cells as well as hippocampal efferent axons. These mice provide a genetic and anatomic tool for analyzing development and reorganization in the hippocampus.     

Treadmill exercise suppresses ischemia-induced increment in apoptosis and cell proliferation in hippocampal dentate gyrus of gerbils

Cerebral ischemia resulting from transient or permanent occlusion of cerebral arteries leads to neuronal cell death and eventually causes neurological impairments. In the present study, the effects of treadmill exercise on apoptosis and cell proliferation in the hippocampal dentate gyrus following transient global ischemia in gerbils were investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunohistochemistry for caspase-3 and 5-bromo-2'-deoxyuridine (BrdU). It was shown that apoptotic cell death and cell proliferation in the hippocampal dentate gyrus were significantly increased following transient global ischemia in gerbils and that treadmill exercise suppressed the ischemia-induced increase in apoptosis and cell proliferation in the dentate gyrus. The present results suggest that treadmill exercise may protect cells from apoptotic death and aid in recovery from the central nervous system sequelae following stroke.     

Prolactin stimulates precursor cells in the adult mouse hippocampus

In the search for ways to combat degenerative neurological disorders, neurogenesis-stimulating factors are proving to be a promising area of research. In this study, we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay, we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition, direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely, PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly, no deficit in precursor proliferation was observed in vivo, indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice, as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus, indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory.     

Environmental enrichment stimulates neurogenesis in apolipoprotein E3 and neuronal apoptosis in apolipoprotein E4 transgenic mice

Neurodegeneration in Alzheimer's disease (AD) is associated with the activation of neurogenesis. The mechanisms underlying this crosstalk between neuronal death and birth and the extent to which it is affected by genetic risk factors of AD are not known. We employed transgenic mice expressing human apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for AD, or expressing human apoE3 (an AD-benign allele), in order to examine the hypothesis that apoE4 tilts the balance between neurogenesis and neuronal cell death in favor of the latter. The results showed an isoform-specific increase in neurogenesis in the hippocampal dentate gyrus (DG) under standard conditions in apoE4-transgenic mice. Environmental stimulation, which increases neurogenesis in the DG of apoE3-transgenic and wild-type mice, had the opposite effect on the apoE4 mice, where it triggered apoptosis while decreasing hippocampal neurogenesis. These effects were specific to the DG and were not observed in the subventricular zone, where neurogenesis was unaffected by either the apoE genotype or the environmental conditions. These in vivo findings demonstrate a linkage between neuronal apoptosis and the impaired neuronal plasticity and cognition of apoE4-transgenic mice, and suggest that similar interactions between apoE4 and environmental factors might occur in AD.     

Wnt signaling in eye organogenesis

The vertebrate eye consists of multiple tissues with distinct embryonic origins. To ensure formation of the eye as a functional organ, development of ocular tissues must be precisely coordinated. Besides intrinsic regulators, several extracellular pathways have been shown to participate in controlling critical steps during eye development. Many components of Wnt/Frizzled signaling pathways are expressed in developing ocular tissues, and substantial progress has been made in the past few years in understanding their function during vertebrate eye development. Here, I summarize recent work using functional experiments to elucidate the roles of Wnt/Frizzled pathways during development of ocular tissues in different vertebrates.Key words: eye, retina, ciliary body, lens, vasculature, Wnt, frizzled, mouse, frog, chick, zebrafish     

Investigating Tonic Wnt Signaling Throughout the Adult CNS and in the Hippocampal Neurogenic Niche of BatGal and Ins-TopGal Mice

Wnt/β-catenin signaling has a well-established role in the development of the central nervous system (CNS), and recent evidence is extending this role to include the regulation of adult hippocampal function, including neurogenesis within the dentate gyrus. While the neuroanatomical expression pattern of many canonical Wnt signaling components have been investigated, the sites of signal integration and functional downstream β-catenin activation remain comparatively less characterized in the adult CNS. Using two independent transgenic β-catenin-activated LacZ reporter mouse lines (BatGal and ins-TopGal), we demonstrate that Wnt/β-catenin signaling is active in discrete regions of the adult mouse CNS. Intriguingly, BatGal mice exhibit a broad pattern of reporter expression in the CNS, while expression in ins-TopGal mice is more restricted. Further investigation of these two lines reveals temporal differences in β-catenin-activated reporter expression during neurogenesis within the adult hippocampus. Ins-TopGal mice display peaks of Wnt/β-catenin-activated reporter expression during early and later stages of neurogenesis suggesting Wnt/β-catenin signaling plays an important role during both progenitor cell amplification as well as neuronal maturation, integration, and/or maintenance; however, results from BatGal mice are not as convincing. Thus our data using ins-TopGal mice are consistent with the idea that Wnt signaling plays diverse roles during adult hippocampal neurogenesis and support the idea that multiple transgenic reporter lines must be rigorously compared during scientific investigations.     

The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function.

Members of the Eph family of receptor tyrosine kinases control many aspects of cellular interactions during development, including axon guidance. Here, we demonstrate that EphB2 also regulates postnatal synaptic function in the mammalian CNS. Mice lacking the EphB2 intracellular kinase domain showed wild-type levels of LTP, whereas mice lacking the entire EphB2 receptor had reduced LTP at hippocampal CA1 and dentate gyrus synapses. Synaptic NMDA-mediated current was reduced in dentate granule neurons in EphB2 null mice, as was synaptically localized NR1 as revealed by immunogold localization. Finally, we show that EphB2 is upregulated in hippocampal pyramidal neurons in vitro and in vivo by stimuli known to induce changes in synaptic structure. Together, these data demonstrate that EphB2 plays an important role in regulating synaptic function.