Arabidopsis thaliana NOP10 is required for gametophyte formation

The female gametophyte is crucial for sexual reproduction of higher plants, yet little is known about the molecular mechanisms underlying its development. Here,we report that Arabidopsis thaliana NOP10(AtNOP10) is required for female gametophyte formation. AtNOP10 was expressed predominantly in the seedling and reproductive tissues, including anthers, pollen grains, and ovules.Mutations in AtNOP10 interrupted mitosis of the functional megaspore during early development and prevented polar nuclear fusion in the embryo sacs. AtNOP10 shares a high level of amino acid sequence similarity with Saccharomyces cerevisiae(yeast) NOP10(ScNOP10), an important component of the H/ACA small nucleolar ribonucleoprotein particles(H/ACA sno RNPs) implicated in 18 S r RNA synthesis and r RNA pseudouridylation. Heterologous expression of ScNOP10 complemented the mutant phenotype of Atnop10. Thus, AtNOP10 influences functional megaspore mitosis and polar nuclear fusion during gametophyte formation in Arabidopsis.     

Smad3 is required for enamel biomineralization

Smad3 is an intracellular signaling molecule that mediates the signal from transforming growth factor-beta (TGF-beta) and activin receptors. In this study, we reveal hypomineralized enamel in mice with the targeted deletion of the Smad3 gene. The Smad3 (-/-) mice had chalky white incisor enamel, while the enamel of the wild-type or Smad3 (+/-) mice was yellow-brown. Histological analysis of the undecalcified sections showed that the enamel thickness of the maxillary incisors in the Smad3 (-/-) mice was similar to that of the wild-type and Smad3 (+/-) mice while that the enamel of the maxillary molars in Smad3 (-/-) mice was disrupted in places. Microcomputed tomography (microCT) analysis revealed that the mineralization of the maxillary incisors and mandibular molars in the Smad3 (-/-) mice showed significant reduction in the degree of mineralization when compared to that of the wild-type and Smad3 (+/-) mice. Scanning electron microscopic (SEM) analysis of the mandibular incisors revealed that the enamel surface of the Smad3 (-/-) mice was irregular and disrupted in places and showed images similar to decalcified mature enamel. The histological analysis of the decalcified sections showed that distinct morphological changes in the ameloblasts at the secretory and maturational stages were not observed between the Smad3 (-/-) and Smad3 (+/-) or wild-type mice, while the enamel matrix was observed in the decalcified sections of the mandibular molars in the Smad3 (-/-) mice. These results suggested that Smad3 was required for enamel biomineralization, and TGF-beta and activin signaling might be critical for its process.     

SNX10 is required for osteoclast formation and resorption activity

RANKL-stimulation of osteoclast precursors results in up-regulation of genes involved in the process of differentiation and activation. In this report we describe the expression and functional characterization of Sorting Nexin 10 (snx10). Snx10 belongs to the sorting nexin (SNX) family, a diverse group of proteins with a common feature: the PX domain, which is involved in membrane trafficking and cargo sorting in endosomes. Snx10 is strongly up-regulated during RANKL-induced osteoclast differentiation in vitro and expressed in osteoclasts in vivo. qPCR analysis confirmed a significant increase in the expression of snx10 in in vitro-derived osteoclasts, as well as in femur and calvaria. Immunohistochemical analysis of mouse embryo sections showed expression in long bone, calvariae, and developing teeth. The expression was limited to cells that also expressed TRAP, demonstrating osteoclastic localization. Confocal immunofluorescence and subcellular fractionation analysis revealed Snx10 localization in the nucleus and in the endoplasmic reticulum (ER). To study a possible role for snx10 in osteoclast differentiation and function we silenced snx10 expression and found that snx10 silencing inhibited RANKL-induced osteoclast formation and osteoclast resorption on hydroxyapatite. Silencing also inhibited TRAP secretion. Taken together, these results confirm that snx10 is expressed in osteoclasts and is required for osteoclast differentiation and activity in vitro. Since inhibition of vesicular trafficking is essential for osteoclast formation and activity and SNX10 is involved in intracellular vesicular trafficking, these studies may identify a new candidate gene involved in the development of human bone diseases including osteoporosis.     

Guidance receptor degradation is required for neuronal connectivity in the Drosophila nervous system

Axon pathfinding and synapse formation rely on precise spatiotemporal localization of guidance receptors. However, little is known about the neuron-specific intracellular trafficking mechanisms that underlie the sorting and activity of these receptors. Here we show that loss of the neuron-specific v-ATPase subunit a1 leads to progressive endosomal guidance receptor accumulations after neuronal differentiation. In the embryo and in adult photoreceptors, these accumulations occur after axon pathfinding and synapse formation is complete. In contrast, receptor missorting occurs sufficiently early in neurons of the adult central nervous system to cause connectivity defects. An increase of guidance receptors, but not of membrane proteins without signaling function, causes specific gain-of-function phenotypes. A point mutant that promotes sorting but prevents degradation reveals spatiotemporally specific guidance receptor turnover and accelerates developmental defects in photoreceptors and embryonic motor neurons. Our findings indicate that a neuron-specific endolysosomal degradation mechanism is part of the cell biological machinery that regulates guidance receptor turnover and signaling.     

Smad7 is required for the development and function of the heart

Transforming growth factor-beta (TGF-beta) family members, including TGF-betas, activins, and bone morphogenetic proteins, exert diverse biological activities in cell proliferation, differentiation, apoptosis, embryonic development, and many other processes. These effects are largely mediated by Smad proteins. Smad7 is a negative regulator for the signaling of TGF-beta family members. Dysregulation of Smad7 is associated with pathogenesis of a variety of human diseases. However, the in vivo physiological roles of Smad7 have not been elucidated due to the lack of a mouse model with significant loss of Smad7 function. Here we report generation and initial characterization of Smad7 mutant mice with targeted deletion of the indispensable MH2 domain. The majority of Smad7 mutant mice died in utero due to multiple defects in cardiovascular development, including ventricular septal defect and non-compaction, as well as outflow tract malformation. The surviving adult Smad7 mutant mice had impaired cardiac functions and severe arrhythmia. Further analyses suggest that Smad2/3 phosphorylation was elevated in atrioventricular cushion in the heart of Smad7 mutant mice, accompanied by increased apoptosis in this region. Taken together, these observations pinpoint an important role of Smad7 in the development and function of the mouse heart in vivo.     

Mammalian E4 is required for cardiac development and maintenance of the nervous system

Ubiquitin conjugation typically requires three classes of enzyme: E1, E2, and E3. A fourth type of enzyme (E4), however, was recently shown to be required for the degradation of certain types of substrate in yeast. We previously identified UFD2a (also known as E4B) as an E4 in mammals. UFD2a is exclusively expressed in cardiac muscle during mouse embryonic development, but it is abundant in neurons of adult mice and is implicated in the pathogenesis of neurodegenerative disease. The precise physiological function of this enzyme has remained largely unknown, however. Here, we show that mice lacking UFD2a die in utero, manifesting marked apoptosis in the developing heart. Polyubiquitylation activity for an E4 substrate was greatly reduced in Ufd2a(-/-) mouse embryonic fibroblasts. Furthermore, Ufd2a(+/-) mice displayed axonal dystrophy in the nucleus gracilis, as well as degeneration of Purkinje cells accompanied by endoplasmic reticulum stress. These animals also developed a neurological disorder. UFD2a thus appears to be essential for the development of cardiac muscle, as well as for the protection of spinocerebellar neurons from degeneration induced by endoplasmic reticulum stress.     

Glycosylphosphatidylinositol-anchored ceruloplasmin is required for iron efflux from cells in the central nervous system

Ceruloplasmin (Cp) is a ferroxidase that converts highly toxic ferrous iron to its non-toxic ferric form. A glycosylphosphatidylinositol (GPI)-anchored form of this enzyme is expressed by astrocytes in the mammalian central nervous system, whereas the secreted form is expressed by the liver and found in serum. Lack of this enzyme results in iron accumulation in the brain and neurodegeneration. Herein, we show using astrocytes purified from the central nervous system of Cp-null mice that GPI-Cp is essential for iron efflux and not involved in regulating iron influx. We also show that GPI-Cp colocalizes on the astrocyte cell surface with the divalent metal transporter IREG1 and is physically associated with IREG1. In addition, IREG1 alone is unable to efflux iron from astrocytes in the absence of GPI-Cp or secreted Cp. We also provide evidence that the divalent metal influx transporter DMT1 is expressed by astrocytes and is likely to mediate iron influx into these glial cells. The coordinated actions of GPI-Cp and IREG1 may be required for iron efflux from neural cells, and disruption of this balance could lead to iron accumulation in the central nervous system and neurodegeneration.     

Smad5 is required for mouse primordial germ cell development

Smad5, together with Smad1 and Smad8, have been implicated as downstream signal mediators for several bone morphogenetic proteins (BMPs). Recent studies have shown that primordial germ cells (PGCs) are absent or greatly reduced in Bmp4 or Bmp8b mutant mice. To define the role of Smad5 in PGC development, we examined PGC number in Smad5 mutant mice by Oct4 whole-mount in situ hybridization and alkaline phosphatase staining. We found ectopic PGC-like cells in the amnion of some Smad5 mutant mice, however, the total number of PGCs was greatly reduced or completely absent in Smad5 mutant embryos, similar to Bmp4 or Bmp8b mutant embryos. Therefore, Smad5 is an important factor involved in PGC generation and localization.     

Smad4 is required for the normal organization of the cartilage growth plate

Smad4 is the central intracellular mediator of transforming growth factor-beta (TGF-beta) signals. To study the role of Smad4 in skeletal development, we introduced a conditional mutation of the gene in chondrocytes using Cre--loxP system. We showed that Smad4 was expressed strongly in prehypertrophic and hypertrophic chondrocytes. The abrogation of Smad4 in chondrocytes resulted in dwarfism with a severely disorganized growth plate characterized by expanded resting zone of chondrocytes, reduced chondrocyte proliferation, accelerated hypertrophic differentiation, increased apoptosis and ectopic bone collars in perichondrium. Meanwhile, Smad4 mutant mice exhibited decreased expression of molecules in Indian hedgehog/parathyroid hormone-related protein (Ihh/PTHrP) signaling. The cultured mutant metatarsal bones failed to response to TGF-beta1, while the hypertrophic differentiation was largely inhibited by Sonic hedgehog (Shh). This indicated that Ihh/PTHrP inhibited the hypertrophic differentiation of chondrocytes independent of the Smad4-mediated TGF-beta signals. All these data provided the first genetic evidence demonstrating that Smad4-mediated TGF-beta signals inhibit the chondrocyte hypertrophic differentiation, and are required for maintaining the normal organization of chondrocytes in the growth plate.     

AMSH is required to degrade ubiquitinated proteins in the central nervous system

Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. AMSH (associated molecule with the SH3 domain of STAM) is a deubiquitination enzyme that participates in the endosomal sorting of several cell-surface molecules. AMSH impairment results in missorted ubiquitinated cargoes in vitro and severe neurodegeneration in vivo, but it is not known how AMSH deficiency causes neuronal damage in the brain. Here, we demonstrate that AMSH^−/− mice developed ubiquitinated protein accumulations as early as embryonic day 10 (E10), and that severe deposits were present in the brain at postnatal day 8 (P8) and P18. Interestingly, TDP-43 was found to accumulate and colocalize with glial marker-positive cells in the brain. Glutamate receptor and p62 accumulations were also found; these molecules colocalized with ubiquitinated aggregates in the brain. These data suggest that AMSH plays an important role in degrading ubiquitinated proteins and glutamate receptors in vivo. AMSH^−/− mice provide an animal model for neurodegenerative diseases, which are commonly characterized by the generation of proteinaceous aggregates.     

Tension is required for fibripositor formation

Embryonic tendon cells (ETCs) have actin-rich fibripositors that accompany parallel bundles of collagen fibrils in the extracellular matrix. To study fibripositor function, we have developed a three-dimensional cell culture system that promotes and maintains fibripositors. We show that ETCs cultured in fixed-length fibrin gels replace the fibrin during ~6 days in culture with parallel bundles of narrow-diameter collagen fibrils that are uniaxially aligned with fibripositors, thereby generating a tendon-like construct. Fibripositors occurred simultaneously with onset of parallel collagen fibrils. Interestingly, the constructs have a tendon-like crimp. In initial experiments to study the effects of tension, we showed that cutting the constructs resulted in loss of tension, loss of fibripositors and the appearance of immature fibrils with no preferred orientation.     

The kinesin-associated protein UNC-76 is required for axonal transport in the Drosophila nervous system

Kinesin-I is essential for the transport of membrane-bound organelles in neural and nonneural cells. However, the means by which kinesin interacts with its intracellular cargoes, and the means by which kinesin-cargo interactions are regulated in response to cellular transport requirements are not fully understood. The C terminus of the Drosophila kinesin heavy chain (KHC) was used in a two-hybrid screen of a Drosophila cDNA library to identify proteins that bind specifically to the kinesin tail domain. UNC-76 is an evolutionarily conserved cytosolic protein that binds to the tail domain of KHC in two-hybrid and copurification assays, indicating that kinesin and UNC-76 form a stable complex in vivo. Loss of Drosophila Unc-76 function results in locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants, suggesting that UNC-76 is required for kinesin-dependent axonal transport. Unc-76 exhibits dosage-sensitive genetic relationships with Khc and Kinesin light chain mutations, further supporting the hypothesis that UNC-76 and kinesin-I work in a common transport pathway. Given the interaction of FEZ1, the mammalian homolog of UNC-76, with protein kinase Czeta, and the role of FEZ1 in axon outgrowth, we propose that UNC-76 helps integrate kinesin activity in response to transport requirements in axons.     

A Xenopus tribbles orthologue is required for the progression of mitosis and for development of the nervous system

The product of the Drosophila gene tribbles inhibits cell division in the ventral furrow of the embryo and thereby allows the normal prosecution of gastrulation. Cell division is also absent in involuting dorsal mesoderm during gastrulation in Xenopus, and to ask whether the two species employ similar mechanisms to coordinate morphogenesis and the cell cycle, we isolated a putative Xenopus homologue of tribbles which we call Xtrb2. Extensive cDNA cloning identified long and short forms of Xtrb2, termed Xtrb2-L and Xtrb2-S, respectively. Xtrb2 is expressed maternally and in mesoderm and ectoderm at blastula and gastrula stages. Later, it is expressed in dorsal neural tube, eyes, and cephalic neural crest. Time-lapse imaging of GFP-tagged Xtrb2-L suggests that during cell division, it is associated with mitotic spindles. Knockdown of Xtrb2 by antisense morpholino oligonucleotides (MOs) disrupted synchronous cell divisions during blastula stages, apparently as a result of delayed progression through mitosis and cytokinesis. At later stages, tissues expressing the highest levels of Xtrb2 were most markedly affected by morpholino knockdown, with perturbation of neural crest and eye development.     

Colonic epithelial expression of ErbB2 is required for postnatal maintenance of the enteric nervous system

We utilized the Cre-LoxP system to establish erbB2 conditional mutant mice in order to investigate the role of erbB2 in postnatal development of the enteric nervous system. The erbB2/nestin-Cre conditional mutants exhibit retarded growth, distended colons, and premature death, resembling human Hirschsprung's disease. Enteric neurons and glia are present at birth in the colon of erbB2/nestin-Cre mutants; however, a marked loss of multiple classes of enteric neurons and glia occurs by 3 weeks of age. Furthermore, we demonstrate that the requirement for erbB2 in maintaining the enteric nervous system is not cell autonomous, but rather erbB2 signaling in the colonic epithelia is required for the postnatal survival of enteric neurons and glia.     

The Drosophila innexin 7 gap junction protein is required for development of the embryonic nervous system

The Drosophila genome encodes eight members of the innexin family of gap junction proteins. Most of the family members are expressed in complex and overlapping expression patterns during Drosophila development. Functional studies and mutant analysis have been performed for only few of the innexin genes. The authors generated an antibody against Innexin7 and studied its expression and functional role in embryonic development by using transgenic RNA interference (RNAi) lines. The authors found Innexin7 protein expression in all embryonic epithelia from early to late stages of development, including in the developing epidermis and the gastrointestinal tract. In early embryonic stages, the authors observed a nuclear localization of Innexin7, whereas Innexin7 was found in a punctuate pattern in the cytoplasm and at the membrane of most epithelial tissues at later stages of development. During central nervous system (CNS) development, Innexin7 was expressed in cells of the neuroectoderm and the mesectoderm and at later stages of embryogenesis, its expression was largely restricted to a segmental pattern of few glia and neuronal cells derived from the midline precursors. Coimmunostaining experiments showed that Innexin7 is expressed in midline glia, and in two different neuronal cells, the pCC and MP2 neurons, which are pioneer cells for axon guidance. RNAi-mediated knock down was used to gain insight into the embryonic function of innexin7. Down-regulation of innexin7 expression resulted in a severe disruption of embryonic nervous system development. Longitudinal, posterior, and anterior commissures were disrupted and the outgrowth of axon fibers of the ventral nerve cord was aberrant, causing peripheral nervous system defects. The results suggest an essential role for innexin7 for axon guidance and embryonic nervous system development in Drosophila.