Shinya Ohata

Dual Roles of Notch in Regulation of Apically Restricted Mitosis and Apicobasal Polarity of Neuroepithelial Cells

How the mitosis of neuroepithelial stem cells is restricted to the apical ventricular area remains unclear. In zebrafish, the mosaic eyes(rw306) (moe/epb41l5(rw306)) mutation disrupts the interaction between the putative adaptor protein Moe and the apicobasal polarity regulator Crumbs (Crb), and impairs the maintenance of neuroepithelial apicobasal polarity. While Crb interacts directly with Notch and inhibits its activity, Moe reverses this inhibition. In the moe(rw306) hindbrain, Notch activity is significantly reduced, and the number of cells that proliferate basally away from the apical area is increased. Surprisingly, activation of Notch in the moe(rw306) mutant rescues not only the basally localized proliferation but also the aberrant neuroepithelial apicobasal polarity. We present evidence that the Crb⋅Moe complex and Notch play key roles in a positive feedback loop to maintain the apicobasal polarity and the apical-high basal-low gradient of Notch activity in neuroepithelial cells, both of which are essential for their apically restricted mitosis.

Loss of Dishevelleds disrupts planar polarity in ependymal motile cilia and results in hydrocephalus

Defects in ependymal (E) cells, which line the ventricle and generate cerebrospinal fluid flow through ciliary beating, can cause hydrocephalus. Dishevelled genes (Dvls) are essential for Wnt signaling, and Dvl2 has been shown to localize to the rootlet of motile cilia. Using the hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) mouse, we show that compound genetic ablation of Dvls causes hydrocephalus. In hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) mutants, E cells differentiated normally, but the intracellular and intercellular rotational alignments of ependymal motile cilia were disrupted. As a consequence, the fluid flow generated by the hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) E cells was significantly slower than that observed in control mice. Dvls were also required for the proper positioning of motile cilia on the apical surface. Tamoxifen-induced conditional removal of Dvls in adult mice also resulted in defects in intracellular rotational alignment and positioning of ependymal motile cilia. These results suggest that Dvls are continuously required for E cell planar polarity and may prevent hydrocephalus.

Neuroepithelial cells require fucosylated glycans to guide the migration of vagus motor neuron progenitors in the developing zebrafish hindbrain.

The molecular mechanisms by which neurons migrate and accumulate to form the neural layers and nuclei remain unclear. The formation of vagus motor nuclei in zebrafish embryos is an ideal model system in which to address this issue because of the transparency of the embryos and the availability of established genetic and molecular biological techniques. To determine the genes required for the formation of the vagus motor nuclei, we performed N-ethyl-N-nitrosourea-based mutant screening using a zebrafish line that expresses green fluorescent protein in the motor neurons. In wild-type embryos, the vagus motor neuron progenitors are born in the ventral ventricular zone, then migrate tangentially in the dorsolateral direction, forming the nuclei. However, in towhead (twdrw685) mutant embryos, the vagus motor neuron progenitors stray medially away from the normal migratory pathway and fail to stop in the right location. The twdrw685 mutant has a defect in the GDP-mannose 4,6 dehydratase (gmds) gene, which encodes a key enzyme in the fucosylation pathway. Levels of fucosylated glycans were markedly and specifically reduced in twdrw685 mutant embryos. Cell transplantation analysis revealed that GMDS is not essential in the vagus motor neuron progenitors for correct formation of the vagus motor nuclei, but is required in the neuroepithelial cells that surround the progenitors. Together, these findings suggest that fucosylated glycans expressed in neuroepithelial cells are required to guide the migration of vagus motor neuron progenitors.

Stress induces mitochondria-mediated apoptosis independent of SAPK/JNK activation in embryonic stem cells.

SAPK/JNK, which belongs to the family of mitogen-activated protein kinase (MAPK), is activated by many types of cellular stresses or extracellular signals and is involved in embryonic development, immune responses, and cell survival or apoptosis. However, the physiological roles of SAPK/JNK in the signaling of stress-induced apoptosis are still controversial. To evaluate the precise function, SAPK/JNK-inactivated mouse embryonic stem (ES) cells were generated by disrupting genes of the MAPK activators, SEK1 and MKK7. Although SAPK/JNK activation by various stresses was completely abolished in sek1–/– mkk7–/– ES cells, apoptotic responses including DNA fragmentation and caspase 3 activation still occurred normally, which displays a sharp contrast to apaf1–/– ES cells exhibiting profound defects in the mitochondria-dependent apoptosis. These normal apoptotic responses without SAPK/JNK activation were also observed in fibroblasts derived from sek1–/– mkk7–/– ES cells. Instead, interleukin-1β (IL-1β)-induced IL-6 gene expression was greatly suppressed in sek1–/– mkk7–/– fibroblasts. These results clearly show that SAPK/JNK activation is responsible for the inflammatory cytokine-induced gene expression but not essentially required for the mitochondria-dependent apoptosis at least in ES or fibroblast-like cells, which are prototypes of all cell lineages.

Negative regulation of wnt11 expression by Jnk signaling during zebrafish gastrulation

Stress‐induced Sapk/Jnk signaling is involved in cell survival and apoptosis. Recent studies have increased our understanding of the physiological roles of Jnk signaling in embryonic development. However, still unclear is the precise function of Jnk signaling during gastrulation, a critical step in the establishment of the vertebrate body plan. Here we use morpholino‐mediated knockdown of the zebrafish orthologs of the Jnk activators Mkk4 and Mkk7 to examine the effect of Jnk signaling abrogation on early vertebrate embryogenesis. Depletion of zebrafish Mkk4b led to abnormal convergent extension (CE) during gastrulation, whereas Mkk7 morphants exhibited defective somitogenesis. Surprisingly, Mkk4b morphants displayed marked upregulation of wnt11, which is the triggering ligand of CE and stimulates Jnk activation via the non‐canonical Wnt pathway. Conversely, ectopic activation of Jnk signaling by overexpression of an active form of Mkk4b led to wnt11 downregulation. Mosaic lineage tracing studies revealed that Mkk4b‐Jnk signaling suppressed wnt11 expression in a non‐cell‐autonomous manner. These findings provide the first evidence that wnt11 itself is a downstream target of the Jnk cascade in the non‐canonical Wnt pathway. Our work demonstrates that Jnk activation is indispensable for multiple steps during vertebrate body plan formation. Furthermore, non‐canonical Wnt signaling may coordinate vertebrate CE movements by triggering Jnk activation that represses the expression of the CE‐triggering ligand wnt11. J. Cell. Biochem. 110: 1022–1037, 2010.

Roles of Wnt Signaling in the Neurogenic Niche of the Adult Mouse Ventricular–Subventricular Zone

In many animal species, the production of new neurons (neurogenesis) occurs throughout life, in a specialized germinal region called the ventricular–subventricular zone (V-SVZ). In this region, neural stem cells undergo self-renewal and generate neural progenitor cells and new neurons. In the olfactory system, the new neurons migrate rostrally toward the olfactory bulb, where they differentiate into mature interneurons. V-SVZ-derived new neurons can also migrate toward sites of brain injury, where they contribute to neural regeneration. Recent studies indicate that two major branches of the Wnt signaling pathway, the Wnt/β-catenin and Wnt/planar cell polarity pathways, play essential roles in various facets of adult neurogenesis. Here, we review the Wnt signaling-mediated regulation of adult neurogenesis in the V-SVZ under physiological and pathological conditions.

Hematopoiesis-dependent expression of CD44 in murine hepatic progenitor cells

The fetal liver serves as the predominant hematopoietic organ until birth. However, the mechanisms underlying this link between hematopoiesis and hepatogenesis are unclear. Previously, we reported the isolation of a monoclonal antibody (anti-Liv8) that specifically recognizes an antigen (Liv8) present in murine fetal livers at embryonic day 11.5 (E11.5). Liv8 is a cell surface molecule expressed by hematopoietic cells in both fetal liver and adult mouse bone marrow. Here, we report that Liv8 is also transiently expressed by hepatoblasts at E11.5. Using protein purification and mass spectrometry, we have identified Liv8 as the CD44 protein. Interestingly, the expression of Liv8/CD44 in fetal liver was completely lost in AML1(-/-) murine embryos, which lack definitive hematopoiesis. These results show that hepatoblasts change from Liv8/CD44-negative to Liv8/CD44-positive status in a hematopoiesis-dependent manner by E11.5, and indicate that Liv8/CD44 expression is an important link between hematopoiesis and hepatogenesis during fetal liver development.

Daple Coordinates Planar Polarized Microtubule Dynamics in Ependymal Cells and Contributes to Hydrocephalus

Motile cilia in ependymal cells, which line the cerebral ventricles, exhibit a coordinated beating motion that drives directional cerebrospinal fluid (CSF) flow and guides neuroblast migration. At the apical cortex of these multi-ciliated cells, asymmetric localization of planar cell polarity (PCP) proteins is required for the planar polarization of microtubule dynamics, which coordinates cilia orientation. Daple is a disheveled-associating protein that controls the non-canonical Wnt signaling pathway and cell motility. Here, we show that Daple-deficient mice present hydrocephalus and their ependymal cilia lack coordinated orientation. Daple regulates microtubule dynamics at the anterior side of ependymal cells, which in turn orients the cilial basal bodies required for the directional cerebrospinal fluid flow. These results demonstrate an important role for Daple in planar polarity in motile cilia and provide a framework for understanding the mechanisms and functions of planar polarization in the ependymal cells.

Arl8b is required for lysosomal degradation of maternal proteins in the visceral yolk sac endoderm of mouse embryos

ABSTRACT The small GTPase Arl8b localizes primarily to lysosomes and is involved in lysosomal motility and fusion. Here, we show that Arl8b is required for lysosomal degradation of maternal proteins in the visceral yolk sac endoderm (VYSE), an apical cell layer of the visceral yolk sac, of mouse embryos. The VYSE actively takes up maternal materials from uterine fluid and degrades them in lysosomes to provide breakdown products to the embryo. Arl8b gene-trap mice (Arl8b−/−) displayed decreased early embryo body size. The Arl8b−/− VYSE exhibited defective endocytic trafficking to the lysosome and accumulation of maternal proteins such as albumin and immunoglobulin G in late endocytic organelles. Furthermore, Transthyretin-Cre;Arl8bflox/flox mice in which Arl8b was ablated specifically in the VYSE also showed decreased embryo body size, defects in trafficking to the lysosome and reduction of the free amino acid level in the embryos. Taken together, these results suggest that Arl8b mediates lysosomal degradation of maternal proteins in the VYSE, thereby contributing to mouse embryonic development. Summary: The small GTPase Arl8b, known to be involved in lysosomal function, contributes to mouse embryonic growth by mediating lysosomal degradation of maternal proteins in the visceral yolk sac endoderm.

Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord

Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuroepithelial cells of the zebrafish hindbrain, axonal collapse in cultured murine hippocampal neurons, and maturation of blood vessels in adult mice. However, the role of R-Ras in neural tube formation remains unknown. Using antisense morpholino oligonucleotides (AMOs), we found that in the spinal cord of zebrafish embryos, the lumen was formed bilaterally in rras morphants, whereas it was formed at the midline in control embryos. As AMO can cause off-target effects, we generated rras mutant zebrafish lines using CRISPR/Cas9 technology. Although these rras mutant embryos did not have a bilateral lumen in the spinal cord, the following findings suggest that the phenotype is unlikely due to an off-target effect of rras AMO: 1) The rras morphant phenotype was rescued by an injection of AMO-resistant rras mRNA, and 2) a bilaterally segregated spinal cord was not observed in rras mutant embryos injected with rras AMO. The results suggest that the function of other ras family genes may be redundant in rras mutants. Previous research reported a bilaterally formed lumen in the spinal cord of zebrafish embryos with a mutation in a planar cell polarity (PCP) gene, van gogh-like 2 (vangl2). In the present study, in cultured cells, R-Ras was co-immunoprecipitated with Vangl2 but not with another PCP regulator, Pricke1. Interestingly, the interaction between R-Ras and Vangl2 was stronger in guanine-nucleotide free point mutants of R-Ras than in wild-type or constitutively active (GTP-bound) forms of R-Ras. R-Ras may regulate neural tube formation in cooperation with Vangl2 in the developing zebrafish spinal cord.