Beta-catenin is essential for lamination but not neurogenesis in mouse retinal development

During vertebrate retinal development, the seven retinal cell types differentiate sequentially from a single population of retinal progenitor cells (RPCs) and organize themselves into a distinct laminar structure. The purpose of this study was to determine whether beta-catenin, which functions both as a nuclear effector for the canonical Wnt signaling pathway and as a regulator of cell adhesion, is required for retinal neurogenesis or lamination. We used the Cre-loxP system to either eliminate beta-catenin or to express a constitutively active form during retinal neurogenesis. Eliminating beta-catenin did not affect cell differentiation, but did result in the loss of the radial arrangement of RPCs and caused abnormal migration of differentiated neurons. As a result, the laminar structure was massively disrupted in beta-catenin-null retinas, although all retinal cell types still formed. In contrast to other neural tissues, eliminating beta-catenin did not significantly reduce the proliferation rate of RPCs; likewise, activating beta-catenin ectopically in RPCs did not result in overproliferation, but loss of neural retinal identity. These results indicate that beta-catenin is essential during retinal neurogenesis as a regulator of cell adhesion but not as a nuclear effector of the canonical Wnt signaling pathway. The results further imply that retinal lamination and retinal cell differentiation are genetically separable processes.     

Varicella-zoster virus infects human embryonic stem cell-derived neurons and neurospheres but not pluripotent embryonic stem cells or early progenitors

Pluripotent human stem cells are a powerful tool for the generation of differentiated cells that can be used for the study of human disease. We recently demonstrated that neurons derived from pluripotent human embryonic stem cells (hESC) can be infected by the highly host-restricted human alphaherpesvirus varicella-zoster virus (VZV), permitting the interaction of VZV with neurons to be readily evaluated in culture. In the present study, we examine whether pluripotent hESC and neural progenitors at intermediate stages of differentiation are permissive for VZV infection. We demonstrate here that VZV infection is blocked in naïve hESC. A block to VZV replication is also seen when a bacterial artificial chromosome (BAC) containing the VZV genome is transfected into hESC. In contrast, related alphaherpesviruses herpes simplex virus 1 (HSV-1) and pseudorabies virus (PrV) productively infect naïve hESC in a cell-free manner, and PrV replicates from a BAC transfected into hESC. Neurons differentiate from hESC via neural progenitor intermediates, as is the case in the embryo. The first in vitro stage at which permissiveness of hESC-derived neural precursors to VZV replication is observed is upon formation of “neurospheres,” immediately after detachment from the inductive stromal feeder layer. These findings suggest that hESC may be useful in deciphering the yet enigmatic mechanisms of specificity of VZV infection and replication.     

Sphingolipids are essential for differentiation but not growth in Leishmania

Sphingolipids (SLs) play critical roles in eukaryotic cells in the formation of lipid rafts, membrane trafficking, and signal transduction. Here we created a SL null mutant in the protozoan parasite Leishmania major through targeted deletion of the key de novo biosynthetic enzyme serine palmitoyltransferase subunit 2 (SPT2). Although SLs are typically essential, spt2- Leishmania were viable, yet were completely deficient in de novo sphingolipid synthesis, and lacked inositol phosphorylceramides and other SLs. Remarkably, spt2- parasites maintained 'lipid rafts' as defined by Triton X-100 detergent resistant membrane formation. Upon entry to stationary phase spt2- failed to differentiate to infective metacyclic parasites and died instead. Death occurred not by apoptosis or changes in metacyclic gene expression, but from catastrophic problems leading to accumulation of small vesicles characteristic of the multivesicular body/multivesicular tubule network. Stage specificity may reflect changes in membrane structure as well as elevated demands in vesicular trafficking required for parasite remodeling during differentiation. We suggest that SL-deficient Leishmania provide a useful biological setting for tests of essential SL enzymes in other organisms where SL perturbation is lethal.     

Widespread tangential dispersion and extensive cell death during early neurogenesis in the mouse neocortex

The development of the mammalian neocortex requires radial and tangential migration of cells. Radial migration of differentiated neurons from the ventricular zone (VZ) is well established. It is hypothesised that an earlier phase of tangential migration of mitotically active cells lays down a widespread periodically spaced set of progenitors that generate radial arrays of postmitotic neurons. We use a transgenic cell lineage marker to label and observe the behaviour of progenitors before and during the early stages of neurogenesis. Using optical projection tomography (OPT), we show that individual progenitor cells generate many radially arrayed columns of periodically spaced cells. Column positions indicate the paths taken by these progenitor cells as they migrate, often over long distances, through the proliferative zone. Clonally related cells can be distributed in both hemispheres, suggesting progenitor cells cross the midline in the anterior neural plate. We observe a dramatic and rapid decline in the number of labelled clones after E13.5, indicating that there is extensive cell death at this time.     

Intraflagellar transport genes are essential for differentiation and survival of vertebrate sensory neurons

Cilia play diverse roles in vertebrate and invertebrate sensory neurons. We show that a mutation of the zebrafish oval (ovl) locus affects a component of the ciliary transport (IFT) mechanism, the IFT88 polypeptide. In mutant retina, cilia are generated but not maintained, producing the absence of photoreceptor outer segments. A loss of cilia also occurs in auditory hair cells and olfactory sensory neurons. In all three sense organs, cilia defects are followed by degeneration of sensory cells. Similar phenotypes are induced by the absence of the IFT complex B polypeptides, ift52 and ift57, but not by the loss of complex A protein, ift140. The degeneration of mutant photoreceptor cells is caused, at least partially, by the ectopic accumulation of opsins. These studies reveal an essential role for IFT genes in vertebrate sensory neurons and implicate the molecular components of intraflagellar transport in degenerative disorders of these cells.     

Tankyrase 1 and tankyrase 2 are essential but redundant for mouse embryonic development

Tankyrases are proteins with poly(ADP-ribose) polymerase activity. Human tankyrases post-translationally modify multiple proteins involved in processes including maintenance of telomere length, sister telomere association, and trafficking of glut4-containing vesicles. To date, however, little is known about in vivo functions for tankyrases. We recently reported that body size was significantly reduced in mice deficient for tankyrase 2, but that these mice otherwise appeared developmentally normal. In the present study, we report generation of tankyrase 1-deficient and tankyrase 1 and 2 double-deficient mice, and use of these mutant strains to systematically assess candidate functions of tankyrase 1 and tankyrase 2 in vivo. No defects were observed in development, telomere length maintenance, or cell cycle regulation in tankyrase 1 or tankyrase 2 knockout mice. In contrast to viability and normal development of mice singly deficient in either tankyrase, deficiency in both tankyrase 1 and tankyrase 2 results in embryonic lethality by day 10, indicating that there is substantial redundancy between tankyrase 1 and tankyrase 2, but that tankyrase function is essential for embryonic development.     

Vitamin E is essential for mouse placentation but not for embryonic development itself

Vitamin E (alpha-tocopherol) was discovered 80 years ago to be an indispensable nutrient for reproduction in the female. However, it has not been clarified when or where vitamin E is required during pregnancy. We examined the role of alpha-tocopherol in pregnancy using alpha-tocopherol transfer protein (Ttpa)-deficient mice fed specific alpha-tocopherol diets that led to daily, measurable change in plasma alpha-tocopherol levels from nearly normal to almost undetectable levels. A dietary supplement of alpha-tocopherol to pregnant Ttpa-/- (homozygous null) mice was shown to be essential for maintenance of pregnancy from 6.5 to 13.5 days postcoitum but found not to be crucial before or after this time span, which corresponds to initial development and maturation of the placenta. In addition, exposure to a low alpha-tocopherol environment after initiation of placental formation might result in necrosis of placental syncytiotrophoblast cells, followed by necrosis of fetal blood vessel endothelial cells. When Ttpa(-/-)-fertilized eggs were transferred into Ttpa+/+ (wild-type) recipients, plasma alpha-tocopherol concentrations in the Ttpa-/- fetuses were below the detection limit but the fetuses grew normally. These results indicate that alpha-tocopherol is indispensable for the proliferation and/or function of the placenta but not necessary for development of the embryo itself.     

Mouse embryonic stem cells are not susceptible to cytomegalovirus but acquire susceptibility during differentiation

BACKGROUND: Cytomegalovirus (CMV) is the most significant infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection in humans. The timing of infection and the susceptibility of cells in early gestational stages are not well understood. In this study we investigated the susceptibility of embryonic stem (ES) cells to CMV infection during differentiation. METHODS: ES cell lines were established from transgenic mice integrated with the murine CMV (MCMV) immediate-early (IE) promoter connected with a reporter lacZ gene. The susceptibility of the ES cells was analyzed in terms of viral gene expression and viral replication after induction of differentiation. RESULTS: ES cells were nonpermissive to MCMV infection in the undifferentiated state. Upon differentiation, permissive cells appeared approximately 2 weeks after the leukemia inhibitory factor was removed. Upon neural differentiation by retinoic acid (RA), glial cells showed specific susceptibility in terms of expression of the viral antigen. The MCMV IE promoter was not activated in ES cells from the transgenic mice. Activation of the IE promoter was detected approximately 2 weeks after induction of differentiation and observed predominantly in glial cells. Upon MCMV infection of the ES cells, viral infection was correlated with the activation of the IE promoter. CONCLUSIONS: ES cells are nonpermissive to MCMV infection and acquire permissiveness about 2 weeks after induction of differentiation, especially in glial cells. Acquisition of permissiveness in differentiated ES cells may be associated with activation of the IE promoter.     

Ubiquinone is necessary for mouse embryonic development but is not essential for mitochondrial respiration

Ubiquinone (UQ) is a lipid found in most biological membranes and is a co-factor in many redox processes including the mitochondrial respiratory chain. UQ has been implicated in protection from oxidative stress and in the aging process. Consequently, it is used as a dietary supplement and to treat mitochondrial diseases. Mutants of the clk-1 gene of the nematode Caenorhabditis elegans are fertile and have an increased life span, although they do not produce UQ but instead accumulate a biosynthetic intermediate, demethoxyubiquinone (DMQ). DMQ appears capable to partially replace UQ for respiration in vivo and in vitro. We have produced a vertebrate model of cells and tissues devoid of UQ by generating a knockout mutation of the murine orthologue of clk-1 (mclk1). We find that mclk1-/- embryonic stem cells and embryos accumulate DMQ instead of UQ. As in the nematode mutant, the activity of the mitochondrial respiratory chain of -/- embryonic stem cells is only mildly affected (65% of wild-type oxygen consumption). However, mclk1-/- embryos arrest development at midgestation, although earlier developmental stages appear normal. These findings indicate that UQ is necessary for vertebrate embryonic development but suggest that mitochondrial respiration is not the function for which UQ is essential when DMQ is present.     

Differentiation of rhesus embryonic stem cells to neural progenitors and neurons

Embryonic stem (ES) cells are pluripotent cells capable of differentiating into cell lineages derived from all primary germ layers including neural cells. In this study we describe an efficient method for differentiating rhesus monkey ES cells to neural lineages and the subsequent isolation of an enriched population of Nestin and Musashi positive neural progenitor (NP) cells. Upon differentiation, these cells exhibit electrophysiological characteristics resembling cultured primary neurons. Embryoid bodies (EBs) were formed in ES growth medium supplemented with 50% MEDII. After 7 days in suspension culture, EBs were transferred to adherent culture and either differentiated in serum containing medium or expanded in serum free medium. Immunocytochemistry on differentiating cells derived from EBs revealed large networks of MAP-2 and NF200 positive neurons. DAPI staining showed that the center of the MEDII-treated EBs was filled with rosettes. NPs isolated from adherent EB cultures expanded in serum free medium were passaged and maintained in an undifferentiated state by culture in serum free N2 with 50% MEDII and bFGF. Differentiating neurons derived from NPs fired action potentials in response to depolarizing current injection and expressed functional ionotropic receptors for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). NPs derived in this way could serve as models for cellular replacement therapy in primate models of neurodegenerative disease, a source of neural cells for toxicity and drug testing, and as a model of the developing primate nervous system.     

Fatty acids but not dexamethasone are essential inducers for chick adipocyte differentiation in vitro

The present study was carried out to clarify the direct effect of fatty acids (FAs) on chick (Gallus gallus) adipocyte differentiation in the absence of dexmethasone (DEX), a commonly used as strong inducer for adipocyte differentiation. Adipocyte differentiation was initiated by maintaining confluent cell in serum-free medium supplemented with FAs. Upon exposure to FAs, glycerol-3-phosphate dehydrogenase activity (GPDH) as adipocyte differentiation marker rapidly increased, and was significantly higher in chick adipocyte than in control cell. The morphology of the FAs-treated cell changed from fibroblast-like to polygon, and the cells accumulated many cytoplasmic lipid droplets as estimated by Oil red O staining. Neither insulin nor bovine serum albumin, as substitutes for serum, had an effect on chick adipocyte differentiation. The FAs-treated cell had a higher protein and mRNA expression levels for peroxisome proliferator-activated receptor-γ (PPARγ), a master regulator of differentiation, compared with untreated cell. In FAs-treated cell, the mRNA expression levels of adipocyte-specific genes, such as CCAAT/enhancer binding protein-α (C/EBP α) and adipocyte fatty acid-binding protein (aP2) were higher than in control cell. These results indicated that FAs, but not DEX, are essential inducers for chick adipocyte differentiation by elevating PPARγ expression.     

Elevation in phosphatidylethanolamine is an early but not essential event for cardiac cell differentiation

The biosynthesis of phosphatidylethanolamine was examined during differentiation of P19 teratocarcinoma cells into cardiac myocytes. P19 cells were induced to undergo differentiation into cardiac myocytes by the addition of dimethyl sulfoxide to the medium. Immunofluorescence labeling confirmed the expression of striated myosin 10 days postinduction of differentiation. The content of phosphatidylethanolamine increased significantly within the first 2 days of differentiation. [1,3-(3)H]Glycerol incorporation into phosphatidylethanolamine was increased 7.2-fold during differentiation, indicating an elevation in de novo synthesis from 1, 2-diacyl-sn-glycerol. The mechanism for the increase in phosphatidylethanolamine levels during cardiac cell differentiation was a 2.8-fold increase in the activity of ethanolaminephosphotransferase, the 1,2-diacyl-sn-glycerol utilizing reaction of the cytidine 5'-diphosphate-ethanolamine pathway of phosphatidylethanolamine biosynthesis. Incubation of P19 cells with the phosphatidylethanolamine biosynthesis inhibitor 8-(4-chlorophenylthio)-cAMP inhibited the differentiation-induced elevation in phosphatidylethanolamine levels but did not affect the expression of striated myosin. The results suggest that elevation in phosphatidylethanolamine is an early event of P19 cell differentiation into cardiac myocytes, but is not essential for differentiation to proceed.     

Jak2 and Tyk2 are necessary for lineage-specific differentiation, but not for the maintenance of self-renewal of mouse embryonic stem cells

As the LIF-induced Jak1/STAT3 pathway has been reported to play a crucial role in self-renewal of mESCs, we sought to determine if Jak2, which is also expressed in mESCs, might also be involved in the pathway. By employing an RNAi strategy, we established both Jak2 and Jak2/Tyk2 knockdown mESC clones. Both Jak2 and Jak2/Tyk2 knockdown clones maintained the undifferentiated state as wild-type controls, even in a very low concentration of LIF. However, we observed not only faster onset of differentiation but also differential expression of tissue-specific lineage genes for ectodermal and mesodermal, but not endodermal origins from embryoid bodies generated from both types of knockdown clones compared to the wild-type. Furthermore, the reduced level of Jak2 caused differentiation of mESCs in the presence of LIF when the Wnt pathway was activated by LiCl treatment. Taken together, we demonstrated that Jak2 and Tyk2 are not involved in LIF-induced STAT3 pathway for self-renewal of mESCs, but play a role in early lineage decision of mESCs to various differentiated cell types.     

L2dtl is essential for cell survival and nuclear division in early mouse embryonic development

l(2)dtl (lethal (2) denticleless), is an embryonic lethal homozygous mutation initially identified in Drosophila melanogaster that produces embryos that lack ventral denticle belts. In addition to nucleotide sequence, bioinformatic analysis has revealed a conservation of critical functional motifs among the human L2DTL, mouse L2dtl, and Drosophila l(2)dtl proteins. The function of the L2DTL protein in the development of mammalian embryos was studied using targeted disruption of the L2dtl gene in mice. The knock-out resulted in early embryonic lethality. L2dtl-/- embryos were deformed and terminated development at the 4-8-cell stage. Microinjection of a small interfering RNA (siRNA) vector (siRNA-L2dtl) into the two-cell stage nuclei of wild-type mouse embryos led to cell cycle progression failure, termination of cell division, and, eventually, embryonic death during the preimplantation stage. Morphological studies of the embryos 54 h after injection showed fragmentation of mitotic chromosomes and chromosomal lagging, hallmarks of mitotic catastrophe. The siRNA-L2dtl-treated embryos eventually lysed and failed to develop into blastocysts after 72 h of in vitro culturing. However, the embryos developed normally after they were microinjected into one nucleus of the two-celled embryos. The siRNA studies in HeLa cells showed that L2dtl protein depletion results in multinucleation and down-regulation of phosphatidylinositol 3-kinase, proliferating cell nuclear antigen, and PTTG1/securin, which might partially explain the mitotic catastrophe observed in L2dtl-depleted mouse embryos. Based on these findings, we conclude that L2dtl gene expression is essential for very early mouse embryonic development.