Zinc finger genes Fezf1 and Fezf2 control neuronal differentiation by repressing Hes5 expression in the forebrain.

Precise control of neuronal differentiation is necessary for generation of a variety of neurons in the forebrain. However, little is known about transcriptional cascades, which initiate forebrain neurogenesis. Here we show that zinc finger genes Fezf1 and Fezf2, which encode transcriptional repressors, are expressed in the early neural stem (progenitor) cells and control neurogenesis in mouse dorsal telencephalon. Fezf1- and Fezf2-deficient forebrains display upregulation of Hes5 and downregulation of neurogenin 2, which is known to be negatively regulated by Hes5. We show that FEZF1 and FEZF2 bind to and directly repress the promoter activity of Hes5. In Fezf1- and Fezf2-deficient telencephalon, the differentiation of neural stem cells into early-born cortical neurons and intermediate progenitors is impaired. Loss of Hes5 suppresses neurogenesis defects in Fezf1- and Fezf2-deficient telencephalon. Our findings reveal that Fezf1 and Fezf2 control differentiation of neural stem cells by repressing Hes5 and, in turn, by derepressing neurogenin 2 in the forebrain.

Atypical protein kinase C regulates primary dendrite specification of cerebellar Purkinje cells by localizing Golgi apparatus.

Neurons have highly polarized structures that determine what parts of the soma elaborate the axon and dendrites. However, little is known about the mechanisms that establish neuronal polarity in vivo. Cerebellar Purkinje cells extend a single primary dendrite from the soma that ramifies into a highly branched dendritic arbor. We used the zebrafish cerebellum to investigate the mechanisms by which Purkinje cells acquire these characteristics. To examine dendritic morphogenesis in individual Purkinje cells, we marked the cell membrane using a Purkinje cell-specific promoter to drive membrane-targeted fluorescent proteins. We found that zebrafish Purkinje cells initially extend multiple neurites from the soma and subsequently retract all but one, which becomes the primary dendrite. In addition, the Golgi apparatus specifically locates to the root of the primary dendrite, and its localization is already established in immature Purkinje cells that have multiple neurites. Inhibiting secretory trafficking through the Golgi apparatus reduces dendritic growth, suggesting that the Golgi apparatus is involved in the dendritic morphogenesis. We also demonstrated that in a mutant of an atypical protein kinase C (aPKC), Prkci, Purkinje cells retain multiple primary dendrites and show disrupted localization of the Golgi apparatus. Furthermore, a mosaic inhibition of Prkci in Purkinje cells recapitulates the aPKC mutant phenotype. These results suggest that the aPKC cell autonomously controls the Golgi localization and thereby regulates the specification of the primary dendrite of Purkinje cells.

Proneural gene-linked neurogenesis in zebrafish cerebellum.

In mammals, cerebellar neurons are categorized as glutamatergic or GABAergic, and are derived from progenitors that express the proneural genes atoh1 or ptf1a, respectively. In zebrafish, three atoh1 genes, atoh1a, atoh1b, and atoh1c, are expressed in overlapping but distinct expression domains in the upper rhombic lip (URL): ptf1a is expressed exclusively in the ventricular zone (VZ). Using transgenic lines expressing fluorescent proteins under the control of the regulatory elements of atoh1a and ptf1a, we traced the lineages of the cerebellar neurons. The atoh1(+) progenitors gave rise not only to granule cells but also to neurons of the anteroventral rhombencephalon. The ptf1a(+) progenitors generated Purkinje cells. The olig2(+) eurydendroid cells, which are glutamatergic, were derived mostly from ptf1a(+) progenitors in the VZ but some originated from the atoh1(+) progenitors in the URL. In the adult cerebellum, atoh1a, atoh1b, and atoh1c are expressed in the molecular layer of the valvula cerebelli and of the medial corpus cerebelli, and ptf1a was detected in the VZ. The proneural gene expression patterns coincided with the sites of proliferating neuronal progenitors in the adult cerebellum. Our data indicate that proneural gene-linked neurogenesis is evolutionarily conserved in the cerebellum among vertebrates, and that the continuously generated neurons help remodel neural circuits in the adult zebrafish cerebellum.

Filopodia formation mediated by receptor tyrosine kinase Ror2 is required for Wnt5a-induced cell migration.

The receptor tyrosine kinase Ror2 plays important roles in developmental morphogenesis. It has recently been shown that Ror2 mediates Wnt5a-induced noncanonical Wnt signaling by activating the Wnt-JNK pathway and inhibiting the beta-catenin-TCF pathway. However, the function of Ror2 in noncanonical Wnt signaling leading to cell migration is largely unknown. We show, using genetically different or manipulated cultured cells, that Ror2 is critical for Wnt5a-induced, but not Wnt3a-induced, cell migration. Ror2-mediated cell migration requires the extracellular cysteine-rich domain (CRD), which is the binding site for Wnt5a, and the cytoplasmic proline-rich domain (PRD) of Ror2. Furthermore, Ror2 can mediate filopodia formation via actin reorganization, irrespective of Wnt5a, and this Ror2-mediated filopodia formation requires the actin-binding protein filamin A, which associates with the PRD of Ror2. Intriguingly, disruption of filopodia formation by suppressing the expression of either Ror2 or filamin A inhibits Wnt5a-induced cell migration, indicating that Ror2-mediated filopodia formation is essential for Wnt5a-induced cell migration.

Anatomy of zebrafish cerebellum and screen for mutations affecting its development.

The cerebellum is important for the integration of sensory perception and motor control, but its structure has mostly been studied in mammals. Here, we describe the cell types and neural tracts of the adult zebrafish cerebellum using molecular markers and transgenic lines. Cerebellar neurons are categorized to two major groups: GABAergic and glutamatergic neurons. The Purkinje cells, which are GABAergic neurons, express parvalbumin7, carbonic anhydrase 8, and aldolase C like (zebrin II). The glutamatergic neurons are vglut1(+) granule cells and vglut2(high) cells, which receive Purkinje cell inputs; some vglut2(high) cells are eurydendroid cells, which are equivalent to the mammalian deep cerebellar nuclei. We found olig2(+) neurons in the adult cerebellum and ascertained that at least some of them are eurydendroid cells. We identified markers for climbing and mossy afferent fibers, efferent fibers, and parallel fibers from granule cells. Furthermore, we found that the cerebellum-like structures in the optic tectum and antero-dorsal hindbrain show similar Parvalbumin7 and Vglut1 expression profiles as the cerebellum. The differentiation of GABAergic and glutamatergic neurons begins 3 days post-fertilization (dpf), and layers are first detectable 5 dpf. Using anti-Parvalbumin7 and Vglut1 antibodies to label Purkinje cells and granule cell axons, respectively, we screened for mutations affecting cerebellar neuronal development and the formation of neural tracts. Our data provide a platform for future studies of zebrafish cerebellar development.

PRMT8 as a phospholipase regulates Purkinje cell dendritic arborization and motor coordination.

The development of vertebrate neurons requires a change in membrane phosphatidylcholine (PC) metabolism. Although PC hydrolysis is essential for enhanced axonal outgrowth mediated by phospholipase D (PLD), less is known about the determinants of PC metabolism on dendritic arborization. We show that protein arginine methyltransferase 8 (PRMT8) acts as a phospholipase that directly hydrolyzes PC, generating choline and phosphatidic acid. We found that PRMT8 knockout mice (prmt8 (-/-)) displayed abnormal motor behaviors, including hindlimb clasping and hyperactivity. Moreover, prmt8 (-/-) mice and TALEN-induced zebrafish prmt8 mutants and morphants showed abnormal phenotypes, including the development of dendritic trees in Purkinje cells and altered cerebellar structure. Choline and acetylcholine levels were significantly decreased, whereas PC levels were increased, in the cerebellum of prmt8 (-/-) mice. Our findings suggest that PRMT8 acts both as an arginine methyltransferase and as a PC-hydrolyzing PLD that is essential for proper neurological functions.

Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis.

The signaling molecule Wnt regulates bone homeostasis through ß-catenin-dependent canonical and ß-catenin-independent noncanonical pathways. Impairment of canonical Wnt signaling causes bone loss in arthritis and osteoporosis; however, it is unclear how noncanonical Wnt signaling regulates bone resorption. Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor (Ror) proteins. We showed that Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhanced osteoclastogenesis. Osteoblast-lineage cells expressed Wnt5a, whereas osteoclast precursors expressed Ror2. Mice deficient in either Wnt5a or Ror2, and those with either osteoclast precursor-specific Ror2 deficiency or osteoblast-lineage cell-specific Wnt5a deficiency showed impaired osteoclastogenesis. Wnt5a-Ror2 signals enhanced receptor activator of nuclear factor-κB (RANK) expression in osteoclast precursors by activating JNK and recruiting c-Jun on the promoter of the gene encoding RANK, thereby enhancing RANK ligand (RANKL)-induced osteoclastogenesis. A soluble form of Ror2 acted as a decoy receptor of Wnt5a and abrogated bone destruction in mouse arthritis models. Our results suggest that the Wnt5a-Ror2 pathway is crucial for osteoclastogenesis in physiological and pathological environments and represents a therapeutic target for bone diseases, including arthritis.

Chk2 kinase is required for methylglyoxal-induced G2/M cell-cycle checkpoint arrest: implication of cell-cycle checkpoint regulation in diabetic oxidative stress signaling.

Methylglyoxal (MG) is a reactive endogenous metabolite that is produced from the process of degradation of triose-phosphates. Under hyperglycemic conditions the rate of MG formation increases as a result of elevated concentrations of precursors. It has been established that MG elicits oxidative stress signaling, leading to the activation of MAP kinases, p38 MAPK and JNK, yet it remains largely unknown about a role of cell-cycle checkpoint regulation in MG-induced signaling. Here, we show that checkpoint kinases, Chk1 and Chk2, as well as their upstream ATM kinase are phosphorylated and activated following MG treatment of cultured cells. This MG-induced activation of Chk1 and Chk2 were inhibited by either aminoguanidine (AG), an inhibitor of production of advanced glycation end products (AGEs) or N-acetyl-l-cysteine (NAC), an anti-oxidant in dose dependent manners, indicating that oxidative stress via AGEs is involved critically in the activation of Chk1 and Chk2 by MG. Furthermore, it was found that cell-cycle synchronized cells exhibited G(2)/M checkpoint arrest following MG treatment, and that siRNA-mediated knock-down of Chk2, but not Chk1, results in a failure of MG-induced G(2)/M arrest. Thus, the results indicate a critical role for Chk2 in MG-induced G(2)/M cell-cycle checkpoint arrest.

The receptor tyrosine kinase Ror2 associates with the melanoma-associated antigen (MAGE) family protein Dlxin-1 and regulates its intracellular distribution.

The mammalian Ror family receptor tyrosine kinases, Ror1 and Ror2, play crucial roles in developmental morphogenesis. Although the functions of Ror1 and Ror2 are redundant, Ror2 exhibits more specific functions during development. We show that when expressed in mammalian cells, Ror2, but not Ror1, associates with the melanoma-associated antigen (MAGE) family protein, Dlxin-1, which is known to bind to the homeodomain proteins Msx2 and Dlx5 and regulate their transcriptional functions. This association requires the cytoplasmic C-terminal region of Ror2, containing proline-rich and serine/threonine-rich domains, and the C-terminal necdin homology domain of Dlxin-1. Interestingly, the cytoplasmic C-terminal region of Ror2 is missing in patients with brachydactyly type B. Interestingly, transient expression and immunohistochemical analyses reveal that both Dlxin-1 and Msx2 are co-localized in the nuclei in the absence of Ror2. In the presence of Ror2, Dlxin-1 is co-localized with Ror2 at the membranous compartments and Msx2 is retained in the nuclei. It was also found that the majority of cellular Dlxin-1 is retained in the membrane fractions of wild-type but not Ror2-/- mouse embryonic fibroblasts. Furthermore, we show that transcriptional activity of Msx2, irrespective of Ror2 kinase activity, is regulated by ectopic expression of Ror2 using a reporter plasmid containing the WIP element. Thus, Ror2 sequesters Dlxin-1 in membranous compartments, thereby affecting the transcriptional function of Msx2.

The receptor tyrosine kinase Ror2 associates with and is activated by casein kinase Iepsilon.

Ror2, a member of the mammalian Ror family of receptor tyrosine kinases, plays important roles in developmental morphogenesis, although the mechanism underlying activation of Ror2 remains largely elusive. We show that when expressed in mammalian cells, Ror2 associates with casein kinase Iepsilon (CKIepsilon), a crucial regulator of Wnt signaling. This association occurs primarily via the cytoplasmic C-terminal proline-rich domain of Ror2. We also show that Ror2 is phosphorylated by CKIepsilon on serine/threonine residues, in its C-terminal serine/threonine-rich 2 domain, resulting in autophosphorylation of Ror2 on tyrosine residues. Furthermore, it was found that association of Ror2 with CKIepsilon is required for its serine/threonine phosphorylation by CKIepsilon. Site-directed mutagenesis of tyrosine residues in Ror2 reveals that the sites of phosphorylation are contained among the five tyrosine residues in the proline-rich domain but not among the four tyrosine residues in the tyrosine kinase domain. Moreover, we show that in mammalian cells, CKIepsilon-mediated phosphorylation of Ror2 on serine/threonine and tyrosine residues is followed by the tyrosine phosphorylation of G protein-coupled receptor kinase 2, a kinase with a developmental expression pattern that is remarkably similar to that of Ror2. Intriguingly, a mutant of Ror2 lacking five tyrosine residues, including the autophosphorylation sites, fails to tyrosine phosphorylate G protein-coupled receptor kinase 2. This indicates that autophosphorylation of Ror2 is required for full activation of its tyrosine kinase activity. These findings demonstrate a novel role for CKIepsilon in the regulation of Ror2 tyrosine kinase.

The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway.

BACKGROUND: Ror2 is an orphan receptor, belonging to the Ror family of receptor tyrosine kinases. Although Ror2 has been shown to play crucial roles in developmental morphogenesis, the precise signalling events that Ror2 mediates remain elusive. Since Ror2 possesses an extracellular cysteine-rich domain (CRD) that resembles the Wnt-binding sites of the Frizzled (Fz) proteins, it is conceivable that Ror2 interacts with members of the Wnt family. RESULTS: Both Ror2-/- and Wnt5a-/- mice exhibit dwarfism, facial abnormalities, short limbs and tails, dysplasia of lungs and genitals, and ventricular septal defects. In vitro binding assay revealed that Wnt5a binds to the CRD of Ror2. Furthermore, Ror2 associates via its CRD with rFz2, a putative receptor for Wnt5a. Interestingly, Wnt5a and Ror2 activate the non-canonical Wnt pathway, as assessed by activation of JNK in cultured cells and inhibition of convergent extension movements in Xenopus. CONCLUSIONS: Our findings indicate that Wnt5a and Ror2 interact physically and functionally. Ror2 may thus act as a receptor for Wnt5a to activate non-canonical Wnt signalling.