Cell death in the CNS of the Wnt-1 mutant mouse

Using the benzothiazolium-4-quinolium dye, TO-PRO-1, to detect cell death in live embryos, we labeled a developmental series of Wnt-1 null mutant and wild type embryos to determine if cell death contributed to the absence of the midbrain and rostral metencephalon observed in Wnt-1 mutant embryos. We found that there is no detectable cell death at early somite stages in Wnt-1 mutant embryos. However, we detected a significant, but transient, population of dying cells within the anterior dorsal metencephalon in 20–29 somite stage embryos. These cells located in the anterior dorsal metencephalon also stain positive using the TUNEL technique that utilizes terminal transferase to label DNA fragments that are typical in the nuclei of apoptotic cells. Thus, programmed cell death plays a role in the loss of the metencephalon, but apparently does not contribute to the earliest aspect of the mutant phenotype, namely the loss of the midbrain. © 1996 John Wiley & Sons, Inc.     

The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain

The Wnt-1 (int-1) proto-oncogene, which encodes a putative signaling molecule, is expressed exclusively in the developing central nervous system and adult testes. To examine the role of Wnt-1, we generated six independent embryonic stem cell lines in which insertion of a neoR gene by homologous recombination inactivated a Wnt-1 allele. Germline chimeras were generated from two lines, and progeny from matings between heterozygous parents were examined. In all day 9.5 fetuses homozygous for mutated Wnt-1 alleles, most of the midbrain and some rostral metencephalon were absent. The remainder of the neural tube and all other tissues were normal. In late-gestation homozygotes, there was virtually no midbrain and no cerebellum, while the rest of the fetus was normal. Homozygotes are born, but die within 24 hr. Thus the normal role of Wnt-1 is in determination or subsequent development of a specific region of the central nervous system.     

Antisense attenuation of Wnt-1 and Wnt-3a expression in whole embryo culture reveals roles for these genes in craniofacial,spinal cord,and cardiac morphogenesis

Wnt-1 and Wnt-3a proto-on-cogenes have been implicated in the development of midbrain and hindbrain structures. Evidence for such a role has been derived from in situ hybridization studies showing Wnt-1 and -3a expression in developing cranial and spinal cord regions and from studies of mutant mice whose Wnt-1 genes have undergone targeted disruption by homologous recombination. Wnt-1 null mutants exhibit cranial defects but no spinal cord abnormalities, despite expression of the gene in these regions. The absence of spinal cord abnormalities is thought to be due to a functional compensation of the Wnt-1 deficiency by related genes, a problem that has complicated the analysis of null mutants of other developmental genes as well. Herein, we describe the attenuation of Wnt-1 expression using antisense oligonucleotide inhibition in mouse embryos grown in culture. We induce similar mid- and hindbrain abnormalities as those seen in the Wnt-1 null mutant mice. Attentuation of Wnt-1 expression was also associated with cardiomegaly resulting in hemostasis. These findings are consistent with the possibility that a subset of Wnt-1 expressing cells include neural crest cells known to contribute to septation of the truncus arteriosus and to formation of the visceral arches. Antisense knockout of Wnt-3a, a gene structurely related to Wnt-1, targeted the forebrain and midbrain region, which were hy-poplastic and failed to expand, and the spinal cord, which exhibited lateral outpocketings at the level of the forelimb buds. Dual antisense knockouts of Wnt-1 and Wnt-3a targeted all brain regions leading to incomplete closure of the cranial neural folds, and an increase in the number and severity of outpocketings along the spinal cord, suggesting that these genes complement one another to produce normal patterning of the spinal cord. The short time required to assess the mutant phenotype (2 days) and the need for limited sequence information of the target gene (20-25 nu-cleotides) make this antisense oligonucleotide/ whole embryo culture system ideal for testing the importance of specific genes and their interactions in murine embryonic development. © 1993 Wiley-Liss, Inc.     

Purified Wnt-5a increases differentiation of midbrain dopaminergic cells and dishevelled phosphorylation

The Wnt family of lipoproteins regulates several aspects of the development of the nervous system. Recently, we reported that Wnt-3a enhances the proliferation of midbrain dopaminergic precursors and that Wnt-5a promotes their differentiation into dopaminergic neurones. Here we report the purification of hemagglutinin-tagged Wnt-5a using a three-step purification method similar to that previously described for Wnt-3a. Haemagglutinin-tagged Wnt-5a was biologically active and induced the differentiation of immature primary midbrain precursors into tyrosine hydroxylase-positive dopaminergic neurones. Using a substantia nigra-derived dopaminergic cell line (SN4741), we found that Wnt-5a, unlike Wnt-3a, did not promote beta-catenin phosphorylation or stabilization. However, both Wnt-5a and Wnt-3a activated dishevelled, as assessed by a phosphorylation-dependent mobility shift. Moreover, the activity of Wnt-5a on dishevelled was blocked by pre-treatment with acyl protein thioesterase-1, indicating that palmitoylation of Wnt-5a is necessary for its function. Thus, our results suggest that Wnt-3a and Wnt-5a, respectively, activate canonical and non-canonical Wnt signalling pathways in ventral midbrain dopaminergic cells. Furthermore, we identify dishevelled as a key player in transducing both Wnt canonical and non-canonical signals in dopaminergic cells.     

Interaction of Wnt-1 proteins with the binding protein BiP.

The mouse Wnt-1 gene, a target for insertional activation in mouse mammary tumor virus-induced mammary tumors, encodes poorly secreted, cysteine-rich glycoproteins required for proper central nervous system development. We have been analyzing the biosynthesis of Wnt-1 proteins in several cell lines that express Wnt-1 cDNA from heterologous promoters. A protein of 78 kDa was found to be associated with the intracellular forms of Wnt-1 proteins in mammalian and avian cells by using multiple antisera against Wnt-1 proteins. We have identified p78 as the binding protein BiP with anti-BiP antisera and by its release from Wnt-1 immunoprecipitates upon incubation with MgCl2 and ATP. Experiments with a Wnt-1 mutant that lacks the sequence encoding the signal peptide indicates that Wnt-1 proteins must enter the secretory pathway in order to interact with BiP. We demonstrate that Wnt-1 proteins are associated with BiP in cells in which active Wnt-1 proteins are produced, such as a cultured mammary epithelial cell line and Wnt-1 transgenic mouse mammary tumor cells. The association of Wnt-1 proteins with BiP may be a factor in determining the efficiency of secretion of Wnt-1 gene products.     

Regional expression of the Wnt-3 gene in the developing mouse forebrain in relationship to diencephalic neuromeres.

During early vertebrate development, a series of neuromeres divides the central nervous system from the forebrain to the spinal cord. Here we examine in more detail the expression of Wnt-3, a member of the Wnt gene family of secreted proteins, in the developing diencephalon, in comparison to the expression of the homeobox gene Dlx-1. In 9.5-day mouse embryos, Wnt-3 is expressed in a restricted area of the diencephalon before any morphological signs of subdivisions appear. Around embryonic day 11.5, Wnt-3 expression becomes restricted to one of the neuromeres of the diencephalon, the dorsal thalamus. Dlx-1 is expressed in a non-overlapping area immediately anterior to and abutting the Wnt-3 expressing domain, corresponding to the ventral thalamus. In addition, Wnt-3 is expressed in the midbrain-hindbrain region. In the adult mouse, Wnt-3 and Dlx-1 are expressed in subsets of neural cells derived from the original areas of expression in the diencephalon. Taken together, our results suggest that Wnt-3 and Dlx-1 provide positional information for the regional specification of neuromeres in the forebrain. The continued expression of these genes in the adult mouse brain suggests a distinct role in the mature CNS.     

Crossregulation between En-2 and Wnt-1 in chick tectal development

En-1, En-2 and Wnt-1 are proposed to be essential signals for the development of the optic tectum in chick embryos. Drosophila engrailed and wingless , homologs of En ( En-1 and En-2 ) and Wnt-1 , respectively, have been shown to crossregulate each other. In the present paper, it is reported that crossregulation between En-2 and Wnt-1 is preserved in the development of the chick optic tectum. When En-2 is overexpressed by the replication competent retroviral vector, Wnt-1 is expressed ectopically at the dorsal midline of the diencephalon. When Wnt-1 is introduced extrinsically either by ectopic transplantation of mesencephalon, or by implantation of Wnt-1 producing cells, En-2 is induced ectopically at the dorsal midline of the tel-diencephalic border. Thus, ectopic expression of En-2 and Wnt-1 leads to crossregulation of each other in the chick brain. As diencephalon transdifferentiates into the optic tectum by an appropriate signal, the crossregulation of En-2 and Wnt-1 in the diencephalon may mimic the relationship required for early development in the tectum.     

过量表达Wnt-1基因诱导P19细胞的神经分化

Ｗｎｔ－１基因在小鼠神经发育过程中起着重要的作用。该基因在胚胎性癌细胞Ｐ１９细胞经分化过程中存在瞬时性表达。利用克隆到的Ｗｎｔ－１基因转染Ｐ１９细胞,可使细胞不经视黄酸诱导,自发向神经细胞方向分化。     

Swaying is a mutant allele of the proto-oncogene Wnt-1

Mice homozygous for the recessive mutation swaying (sw) are characterized by ataxia and hypertonia, attributed to the malformation of anterior regions of the cerebellum. We show that sw is a deletion of a single base pair from the proto-oncogene Wnt-1. The deletion is predicted to cause premature termination of translation, eliminating the carboxy-terminal half of the Wnt-1 protein. Histological examination shows that sw is phenotypically identical to a previously described wnt-1 mutation introduced into mice by gene targeting. Although both mutations in Wnt-1 disrupt primarily the development of the anterior cerebellum, they also exhibit a variability in expressivity such that rostrally adjacent structures in the midbrain and caudally adjacent structures in the posterior cerebellum can also be affected.     

Development of midbrain and anterior hindbrain ocular motoneurons in normal and Wnt-1 knockout mice

The effect of homozygotic Wnt-1^?/? mutations on the development of ocular motoneurons was examined with the lipophilic dye DiI and compared to control and phenotypic wild-type mouse embryos. A piece of DiI-soaked filter paper was inserted into the orbit, the midbrain, or rhombomere 5 of the hindbrain in six paraformaldehydefixed litters (10.5, 12.5, and 14.5 days postcoitum) containing Wnt-1, Wnt^+/?, and Wnt-1^+/+ individuals and three control litters. We labeled all ocular motoneurons retrogradely and all relevant nerves anterogradely in all control and phenotypic wild-type animals. In all phenotypically identified Wnt-1^?/? mutants we could always label the abducens nerve and motoneurons and the optic fibers to the thalamus, but we were unable to lable oculomotor or trochlear nerves or motoneurons. In addition to Wnt-1 knockout mutants, we also labeled mice from the WZT9B transgenic line carrying a lacZ reporter gene driven by the Wnt-1 gene enhancer. In these embryos we tested for co-localization of Wnt-1 expression in biotinylated dextran amine-labeled ocular motoneurons using a newly developed technique. In younger embryos we obtained evidence for co-localization of the β-galactosidase reaction product derived from lacZ gene activity in some retrogradely filled oculomotor motoneurons and adjacent to other oculomotor and the trochlear motoneurons. Acetylcholine esterase, a marker of early differentiating cholinergic neurons, showed a similar topology with respect to the lacZ reaction product. Thus, at least some future oculomotor motoneurons express Wnt-1, whereas others and the trochlear motoneurons caudal to the ventral midbrain expression of Wnt-1 may be exposed to the short range diffusion of the Wnt-1 gene product. Thus, the Wnt-1^?/? mutation precludes formation or survival of midbrain and anterior hindbrain neurons, including oculomotor and trochlear motoneurons. © 1995 John Wiley & Sons, Inc.     

Wnt-5a Ligand Modulates Mitochondrial Fission-Fusion in Rat Hippocampal Neurons

The Wnt signaling pathway plays an important role in developmental processes, including embryonic patterning, cell specification, and cell polarity. Wnt components participate in the development of the central nervous system, and growing evidence indicates that this pathway also regulates the function of the adult nervous system. In this study, we report that Wnt-5a, a noncanonical Wnt ligand, is a potent activator of mitochondrial dynamics and induces acute fission and fusion events in the mitochondria of rat hippocampal neurons. The effect of Wnt-5a was inhibited in the presence of sFRP, a Wnt scavenger. Similarly, the canonical Wnt-3a ligand had no effect on mitochondrial fission-fusion events, suggesting that this effect is specific for Wnt-5a alone. We also show that the Wnt-5a effects on mitochondrial dynamics occur with an increase in both intracellular and mitochondrial calcium (Ca²⁺), which was correlated with an increased phosphorylation of Drp1(Ser-616) and a decrease of Ser-637 phosphorylation, both indicators of mitochondrial dynamics. Electron microscope analysis of hippocampal tissues in the CA1 region showed an increase in the number of mitochondria present in the postsynaptic region, and this finding correlated with a change in mitochondrial morphology. We conclude that Wnt-5a/Ca²⁺ signaling regulates the mitochondrial fission-fusion process in hippocampal neurons, a feature that might help to further understand the role of Wnt-related pathologies, including neurodegenerative diseases associated with mitochondrial dysfunction, and represents a potentially important link between impaired metabolic function and degenerative disorders.     

Region specific gene expressions in the central nervous system of the ascidian embryo

The vertebrate brain is regionalized during development into forebrain, midbrain and hindbrain. Fibroblast growth factor 8 (FGF8) is expressed in the midbrain/hindbrain boundary (MHB) and functions as an organizer molecule. Previous studies demonstrated that the brain of basal chordates or ascidians is also regionalized at least into fore/midbrain and hindbrain. To better understand the ascidian brain regionalization, the expression of the Ciona Fgf8/17/18 gene was compared with the expression of Otx, En and Pax2/5/8 genes. The expression pattern of these genes resembled that of the genes in the vertebrate forebrain, midbrain, MHB and hindbrain, each of those domains being characterized by sole or combined expression of Otx, Pax2/5/8, En and Fgf8/17/18. In addition, the putative forebrain and midbrain expressed Ci-FgfL and Ci-Fgf9/16/20, respectively. Therefore, the regionalization of the ascidian larval central nervous system was also marked by the expression of Fgf genes.     

Region specific gene expressions in the central nervous system of the ascidian embryo

The vertebrate brain is regionalized during development into forebrain, midbrain and hindbrain. Fibroblast growth factor 8 (FGF8) is expressed in the midbrain/hindbrain boundary (MHB) and functions as an organizer molecule. Previous studies demonstrated that the brain of basal chordates or ascidians is also regionalized at least into fore/midbrain and hindbrain. To better understand the ascidian brain regionalization, the expression of the Ciona Fgf8/17/18 gene was compared with the expression of Otx, En and Pax2/5/8 genes. The expression pattern of these genes resembled that of the genes in the vertebrate forebrain, midbrain, MHB and hindbrain, each of those domains being characterized by sole or combined expression of Otx, Pax2/5/8, En and Fgf8/17/18. In addition, the putative forebrain and midbrain expressed Ci-FgfL and Ci-Fgf9/16/20, respectively. Therefore, the regionalization of the ascidian larval central nervous system was also marked by the expression of Fgf genes.