Maki Wakamiya

Requirement for Wnt3 in vertebrate axis formation

Several studies have implicated Wnt signalling in primary axis formation during vertebrate embryogenesis, yet no Wnt protein has been shown to be essential for this process. In the mouse, primitive streak formation is the first overt morphological sign of the anterior-posterior axis. Here we show that Wnt3 is expressed before gastrulation in the proximal epiblast of the egg cylinder, then is restricted to the posterior proximal epiblast and its associated visceral endoderm and subsequently to the primitive streak and mesoderm. Wnt3–/– mice develop a normal egg cylinder but do not form a primitive streak, mesoderm or node. The epiblast continues to proliferate in an undifferentiated state that lacks anterior-posterior neural patterning, but anterior visceral endoderm markers are expressed and correctly positioned. Our results suggest that regional patterning of the visceral endoderm is independent of primitive streak formation, but the subsequent establishment of anterior-posterior neural pattern in the ectoderm is dependent on derivatives of the primitive streak. These studies provide genetic proof for the requirement of Wnt3 in primary axis formation in the mouse.

Loss of Gcn512 leads to increased apoptosis and mesodermal defects during mouse development

Histone acetyltransferases regulate transcription, but little is known about the role of these enzymes in developmental processes. Gcn5 (encoded by Gcn5l2) and Pcaf, mouse histone acetyltransferases, share similar sequences and enzymatic activities. Both interact with p300 and CBP (encoded by Ep300 and Crebbp, respectively), two other histone acetyltransferases that integrate multiple signalling pathways. Pcaf is thought to participate in many of the cellular processes regulated by p300/CBP (refs 2–8), but the functions of Gcn5 are unknown in mammalian cells. Here we show that the gene Pcaf is dispensable in mice. In contrast, Gcn5l2-null embryos die during embryogenesis. These embryos develop normally to 7.5 days post coitum (d.p.c.), but their growth is severely retarded by 8.5 d.p.c. and they fail to form dorsal mesoderm lineages, including chordamesoderm and paraxial mesoderm. Differentiation of extra-embryonic and cardiac mesoderm seems to be unaffected. Loss of the dorsal mesoderm lineages is due to a high incidence of apoptosis in the Gcn5l2 mutants that begins before the onset of morphological abnormality. Embryos null for both Gcn5l2 and Pcaf show even more severe defects, indicating that these histone acetyltransferases have overlapping functions during embryogenesis. Our studies are the first to demonstrate that specific acetyltransferases are required for cell survival and mesoderm formation during mammalian development.

β-Catenin regulates Cripto- and Wnt3-dependent gene expression programs in mouse axis and mesoderm formation

Gene expression profiling of β-catenin, Cripto and Wnt3 mutant mouse embryos has been used to characterise the genetic networks that regulate early embryonic development. We have defined genes whose expression is regulated by β-catenin during formation of the anteroposterior axis and the mesoderm, and have identified Cripto, which encodes a Nodal co-receptor, as a primary target of β-catenin signals both in embryogenesis as well as in colon carcinoma cell lines and tissues. We have also defined groups of genes regulated by Wnt3/β-catenin signalling during primitive streak and mesoderm formation. Our data assign a key role to β-catenin upstream of two distinct gene expression programs during anteroposterior axis and mesoderm formation.

Generation of a Transgenic Mouse Model of Middle East Respiratory Syndrome Coronavirus Infection and Disease

ABSTRACT The emergence of Middle East respiratory syndrome-coronavirus (MERS-CoV) in the Middle East since 2012 has caused more than 900 human infections with ∼40% mortality to date. Animal models are needed for studying pathogenesis and for development of preventive and therapeutic agents against MERS-CoV infection. Nonhuman primates (rhesus macaques and marmosets) are expensive models of limited availability. Although a mouse lung infection model has been described using adenovirus vectors expressing human CD26/dipeptidyl peptidase 4 (DPP4), it is believed that a transgenic mouse model is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. We show that transgenic mice globally expressing hCD26/DPP4 were fully permissive to MERS-CoV infection, resulting in relentless weight loss and death within days postinfection. High infectious virus titers were recovered primarily from the lungs and brains of mice at 2 and 4 days postinfection, respectively, whereas viral RNAs were also detected in the heart, spleen, and intestine, indicating a disseminating viral infection. Infected Tg+ mice developed a progressive pneumonia, characterized by extensive inflammatory infiltration. In contrast, an inconsistent mild perivascular cuffing was the only pathological change associated with the infected brains. Moreover, infected Tg+ mice were able to activate genes encoding for many antiviral and inflammatory mediators within the lungs and brains, coinciding with the high levels of viral replication. This new and unique transgenic mouse model will be useful for furthering knowledge of MERS pathogenesis and for the development of vaccine and treatments against MERS-CoV infection. IMPORTANCE Small and economical animal models are required for the controlled and extensive studies needed for elucidating pathogenesis and development of vaccines and antivirals against MERS. Mice are the most desirable small-animal species for this purpose because of availability and the existence of a thorough knowledge base, particularly of genetics and immunology. The standard small animals, mice, hamsters, and ferrets, all lack the functional MERS-CoV receptor and are not susceptible to infection. So, initial studies were done with nonhuman primates, expensive models of limited availability. A mouse lung infection model was described where a mouse adenovirus was used to transfect lung cells for receptor expression. Nevertheless, all generally agree that a transgenic mouse model expressing the DPP4 receptor is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. This new and unique transgenic mouse model will be useful for furthering MERS research.

A live-attenuated Zika virus vaccine candidate induces sterilizing immunity in mouse models

Zika virus (ZIKV) infection of pregnant women can cause a wide range of congenital abnormalities, including microcephaly, in the infant, a condition now collectively known as congenital ZIKV syndrome. A vaccine to prevent or significantly attenuate viremia in pregnant women who are residents of or travelers to epidemic or endemic regions is needed to avert congenital ZIKV syndrome, and might also help to suppress epidemic transmission. Here we report on a live-attenuated vaccine candidate that contains a 10-nucleotide deletion in the 3′ untranslated region of the ZIKV genome (10-del ZIKV). The 10-del ZIKV is highly attenuated, immunogenic, and protective in type 1 interferon receptor–deficient A129 mice. Crucially, a single dose of 10-del ZIKV induced sterilizing immunity with a saturated neutralizing antibody titer, which no longer increased after challenge with an epidemic ZIKV, and completely prevented viremia. The immunized mice also developed a robust T cell response. Intracranial inoculation of 1-d-old immunocompetent CD-1 mice with 1 × 104 infectious focus units (IFU) of 10-del ZIKV caused no mortality, whereas infections with 10 IFU of wild-type ZIKV were lethal. Mechanistically, the attenuated virulence of 10-del ZIKV may be due to decreased viral RNA synthesis and increased sensitivity to type-1-interferon inhibition. The attenuated 10-del ZIKV was incapable of infecting mosquitoes after oral feeding of spiked-blood meals, representing an additional safety feature. Collectively, the safety and efficacy results suggest that further development of this promising, live-attenuated ZIKV vaccine candidate is warranted.

Inactivation of hnRNP K by expanded intronic AUUCU repeat induces apoptosis via translocation of PKCdelta to mitochondria in spinocerebellar ataxia 10.

We have identified a large expansion of an ATTCT repeat within intron 9 of ATXN10 on chromosome 22q13.31 as the genetic mutation of spinocerebellar ataxia type 10 (SCA10). Our subsequent studies indicated that neither a gain nor a loss of function of ataxin 10 is likely the major pathogenic mechanism of SCA10. Here, using SCA10 cells, and transfected cells and transgenic mouse brain expressing expanded intronic AUUCU repeats as disease models, we show evidence for a key pathogenic molecular mechanism of SCA10. First, we studied the fate of the mutant repeat RNA by in situ hybridization. A Cy3-(AGAAU)10 riboprobe detected expanded AUUCU repeats aggregated in foci in SCA10 cells. Pull-down and co-immunoprecipitation data suggested that expanded AUUCU repeats within the spliced intronic sequence strongly bind to hnRNP K. Co-localization of hnRNP K and the AUUCU repeat aggregates in the transgenic mouse brain and transfected cells confirmed this interaction. To examine the impact of this interaction on hnRNP K function, we performed RT–PCR analysis of a splicing-regulatory target of hnRNP K, and found diminished hnRNP K activity in SCA10 cells. Cells expressing expanded AUUCU repeats underwent apoptosis, which accompanied massive translocation of PKCδ to mitochondria and activation of caspase 3. Importantly, siRNA–mediated hnRNP K deficiency also caused the same apoptotic event in otherwise normal cells, and over-expression of hnRNP K rescued cells expressing expanded AUUCU repeats from apoptosis, suggesting that the loss of function of hnRNP K plays a key role in cell death of SCA10. These results suggest that the expanded AUUCU–repeat in the intronic RNA undergoes normal transcription and splicing, but causes apoptosis via an activation cascade involving a loss of hnRNP K activities, massive translocation of PKCδ to mitochondria, and caspase 3 activation.

The cerberus‐related gene, Cerr1, is not essential for mouse head formation

Summary: The Xenopus cerberus gene encodes a secreted factor expressed in the Spemann organizer that can cause ectopic head formation when its mRNA is injected into Xenopus embryos. In mouse, the cerberus‐related gene, Cerr1, is expressed in the anterior mesendoderm that underlies the presumptive anterior neural plate and its expression is downregulated in Lim1 headless embryos. To determine whether Cerr1 is required for head formation we generated a null mutation in Cerr1 by gene targeting in mouse embryonic stem cells. We found that head formation is normal in Cerr1‐/‐ embryos and we detected no obvious phenotypic defects in adult Cerr1‐/‐ mice. However, in embryonic tissue layer recombination assays, Cerr1‐/‐ presomitic/somitic mesoderm, unlike Cerr1‐expressing wild‐type presomitic/somitic mesoderm, was unable to maintain expression of the anterior neural marker gene Otx2 in ectoderm explants. These findings suggest that establishment of anterior identity in the mouse may involve the action of multiple functionally redundant factors. genesis 26:253–258, 2000.

The role of ataxin 10 in the pathogenesis of spinocerebellar ataxia type 10

Background: Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant disorder characterized by cerebellar ataxia and seizures. SCA10 is caused by an expansion of an ATTCT pentanucleotide repeat in intron 9 of the ataxin 10 (ATXN10) gene encoding an approximately 55-kd protein of unknown function. However, how this mutation leads to SCA10 is unknown. Methods: In an effort to understand the pathogenic mechanism of SCA10, the authors conducted a series of experiments to address the effect of repeat expansion on the transcription and RNA processing of the ATXN10 gene. In addition, we generated Sca10 (mouse ataxin 10 homolog)–null mice and addressed the role of Sca10 gene dosage on the cerebellum. Results: Mutant ATXN10 allele is transcribed at the normal level, and the pre-mRNA containing an expanded repeat is processed normally in patient-derived cells. Sca10-null mice exhibited embryonic lethality. Heterozygous mutants were overtly normal and did not develop SCA10 phenotype Conclusion: A simple gain of function or loss of function of ATXN10 is unlikely to be the major pathogenic mechanism contributing to the spinocerebellar ataxia type 10 phenotype.

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Significance Our studies combining genetic and pharmacological manipulations provide convincing evidence that exchange protein directly activated by cAMP (Epac) 1 plays a critical role in fatal spotted fever group rickettsioses. Inhibition of Epac1 suppresses bacterial adhesion and/or invasion. Most importantly, we show that a small-molecule Epac inhibitor can prevent and suppress rickettsial infection. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses. This is significant from the biodefense viewpoint because it suggests that Epac1 inhibitor can be potentially used as a prophylaxis to thwart initial bacterial infection in the event of a bioterrorism threat. Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.

Characterization and Demonstration of the Value of a Lethal Mouse Model of Middle East Respiratory Syndrome Coronavirus Infection and Disease

ABSTRACT Characterized animal models are needed for studying the pathogenesis of and evaluating medical countermeasures for persisting Middle East respiratory syndrome-coronavirus (MERS-CoV) infections. Here, we further characterized a lethal transgenic mouse model of MERS-CoV infection and disease that globally expresses human CD26 (hCD26)/DPP4. The 50% infectious dose (ID50) and lethal dose (LD50) of virus were estimated to be <1 and 10 TCID50 of MERS-CoV, respectively. Neutralizing antibody developed in the surviving mice from the ID50/LD50 determinations, and all were fully immune to challenge with 100 LD50 of MERS-CoV. The tissue distribution and histopathology in mice challenged with a potential working dose of 10 LD50 of MERS-CoV were subsequently evaluated. In contrast to the overwhelming infection seen in the mice challenged with 105 LD50 of MERS-CoV, we were able to recover infectious virus from these mice only infrequently, although quantitative reverse transcription-PCR (qRT-PCR) tests indicated early and persistent lung infection and delayed occurrence of brain infection. Persistent inflammatory infiltrates were seen in the lungs and brain stems at day 2 and day 6 after infection, respectively. While focal infiltrates were also noted in the liver, definite pathology was not seen in other tissues. Finally, using a receptor binding domain protein vaccine and a MERS-CoV fusion inhibitor, we demonstrated the value of this model for evaluating vaccines and antivirals against MERS. As outcomes of MERS-CoV infection in patients differ greatly, ranging from asymptomatic to overwhelming disease and death, having available both an infection model and a lethal model makes this transgenic mouse model relevant for advancing MERS research. IMPORTANCE Fully characterized animal models are essential for studying pathogenesis and for preclinical screening of vaccines and drugs against MERS-CoV infection and disease. When given a high dose of MERS-CoV, our transgenic mice expressing hCD26/DPP4 viral receptor uniformly succumbed to death within 6 days, making it difficult to evaluate host responses to infection and disease. We further characterized this model by determining both the ID50 and the LD50 of MERS-CoV in order to establish both an infection model and a lethal model for MERS and followed this by investigating the antibody responses and immunity of the mice that survived MERS-CoV infection. Using the estimated LD50 and ID50 data, we dissected the kinetics of viral tissue distribution and pathology in mice challenged with 10 LD50 of virus and utilized the model for preclinical evaluation of a vaccine and drug for treatment of MERS-CoV infection. This further-characterized transgenic mouse model will be useful for advancing MERS research.