Nuclear phosphatase PPM1G in cellular survival and neural development

Background: PPM1G is a nuclear localized serine/threonine phosphatase implicated to be a regulator of chromatin remodeling, mRNA splicing, and DNA damage. However, its in vivo function is unknown. Results: Here we show that ppm1g expression is highly enriched in the central nervous system during mouse and zebrafish development. ppm1g^?/? mice were embryonic lethal with incomplete penetrance after E12.5. Rostral defects, including neural tube and craniofacial defects were observed in ppm1g^?/? embryos associated with increased cell death in the neural epithelium. In zebrafish, loss of ppm1g also led to neural defects with aberrant neural marker gene expression. Primary fibroblasts from ppm1g^?/? embryos failed to grow without immortalization while immortalized ppm1g^?/? fibroblasts had increased cell death upon oxidative and genotoxic stress when compared to wild type fibroblasts. Conclusions: Our in vivo and in vitro studies revealed a critical role for PPM1G in normal development and cell survival. Developmental Dynamics 242:1101–1109, 2013. © 2013 Wiley Periodicals, Inc.     

Tulp3 is a critical repressor of mouse hedgehog signaling

Precise regulation of the morphogen sonic hedgehog (Shh) and modulation of the Shh signaling pathway is required for proper specification of cell fate within the developing limbs and neural tube, and resultant tissue morphogenesis. Tulp3 (tubby‐like protein 3) is a protein of unknown function which has been implicated in nervous system development through gene knockout studies. We demonstrate here that mice lacking the Tulp3 gene develop abnormalities of both the neural tube and limbs consistent with improper regulation of Shh signaling. Tulp3^?/? embryos show expansion of Shh target gene expression and display a ventralization of neural progenitor cells in the caudal neural tube. We further show that Tulp3^?/?/Shh^?/? compound mutant embryos resemble Tulp3 mutants, and express Shh target genes in the neural tube and limbs which are not expressed in Shh^?/? embryos. This work uncovers a novel role for Tulp3 as a negative regulatory factor in the Hh pathway. Developmental Dynamics 238:1140–1149, 2009. © 2009 Wiley‐Liss, Inc.     

Neural stem cell self‐renewal requires the Mrj co‐chaperone

The Mrj co‐chaperone is expressed throughout the mouse conceptus, yet its requirement for placental development has prohibited a full understanding of its embryonic function. Here, we show that Mrj^?/? embryos exhibit neural tube defects independent of the placenta phenotype, including exencephaly and thin‐walled neural tubes. Molecular analyses revealed fewer proliferating cells and a down‐regulation of early neural progenitor (Pax6, Olig2, Hes5) and neuronal (Nscl2, SCG10) cell markers in Mrj^?/? neuroepithelial cells. Furthermore, Mrj^?/? neurospheres are significantly smaller and form fewer secondary neurospheres indicating that Mrj is necessary for self‐renewal of neural stem cells. However, the molecular function of Mrj in this context remains elusive because Mrj does not colocalize with Bmi‐1, a self‐renewal protein. Furthermore, unlike in Mrj^?/? placentas, intermediate filament‐containing aggregates do not accumulate in Mrj^?/? neuroepithelium, ruling out nestin as a substrate for Mrj. Regardless, Mrj plays an important role in neural stem cell self‐renewal. Developmental Dynamics 238:2564–2574, 2009. © 2009 Wiley‐Liss, Inc.     

Loss of the small GTPase Arl8b results in abnormal development of the roof plate in mouse embryos

Lysosomes are acidic organelles responsible for degrading both exogenous and endogenous materials. The small GTPase Arl8 localizes primarily to lysosomes and is involved in lysosomal function. In the present study, using Arl8b gene‐trapped mutant (Arl8b^?/?) mice, we show that Arl8b is required for the development of dorsal structures of the neural tube, including the thalamus and hippocampus. In embryonic day (E) 10.5 Arl8b^?/? embryos, Sox1 (a neuroepithelium marker) was ectopically expressed in the roof plate, whereas the expression of Gdf7 and Msx1 (roof plate markers) was reduced in the dorsal midline of the midbrain. Ectopic expression of Sox1 in Arl8b^?/? embryos was detected also at E9.0 in the neural fold, which gives rise to the roof plate. In addition, the levels of Bmp receptor IA and phosphorylated Smad 1/5/8 (downstream of BMP signaling) were increased in the neural fold of E9.0 Arl8b^?/? embryos. These results suggest that Arl8b is involved in the development of the neural fold and the subsequently formed roof plate, possibly via control of BMP signaling.     

Ror2 is required for midgut elongation during mouse development

The receptor tyrosine kinase Ror2 acts as a receptor for Wnt5a to mediate noncanonical Wnt signaling, and it plays essential roles in morphogenesis. Ror2^?/? embryos exhibit phenotypes similar to, albeit generally milder than, those of Wnt5a^?/? embryos. During mouse embryogenesis, Ror2 is expressed in various organs and regions, although little is known about its expression pattern and roles in the developing gut, while Wnt5a is expressed in the developing gut, where its absence causes abnormal phenotypes. Here, we demonstrated that Ror2 was strongly and differentially expressed in the rostral and middle midgut endoderm from embryonic day (E) 10.5 through embryonic day (E) 12.5. At E11.5, Ror2^?/? embryos exhibited a shorter middle midgut with a larger diameter and more accumulation of epithelial cells in the middle midgut than control embryos, while the total cell numbers remained unaltered. These findings suggest that Ror2 plays important roles in midgut elongation by means of an epithelial convergent extension mechanism. Developmental Dynamics 239:941–953, 2010. © 2010 Wiley‐Liss, Inc.     

Rare Deleterious PARD3 Variants in the aPKC‐Binding Region are Implicated in the Pathogenesis of Human Cranial Neural Tube Defects Via Disrupting Apical Tight Junction Formation

Increasing evidence that mutation of planar cell polarity (PCP) genes contributes to human cranial neural tube defect (NTD) susceptibility prompted us to hypothesize that rare variants of genes in the core apical–basal polarity (ABP) pathway are risk factors for cranial NTDs. In this study, we screened for rare genomic variation of PARD3 in 138 cranial NTD cases and 274 controls. Overall, the rare deleterious variants of PARD3 were significantly associated with increased risk for cranial NTDs (11/138 vs.7/274, P < 0.05, OR = 3.3). These NTD‐specific variants were significantly enriched in the aPKC‐binding region (6/138 vs. 0/274, P < 0.01). The East Asian cohort in the ExAC database and another Chinese normal cohort further supported this association. Over‐expression analysis in HEK293T and MDCK cells confirmed abnormal aPKC binding or interaction for two PARD3 variants (p.P913Q and p.D783G), resulting in defective tight junction formation via disrupted aPKC binding. Functional analysis in human neural progenitor cells and chick embryos revealed that PARD3 knockdown gave rise to abnormal cell polarity and compromised the polarization process of neuroepithelial tissue. Our studies suggest that rare deleterious variants of PARD3 in the aPKC‐binding region contribute to human cranial NTDs, possibly by disrupting apical tight junction formation and subsequent polarization process of the neuroepithelium.     

Generation and characterization of a novel neural crest marker allele,Inka1‐LacZ,reveals a role for Inka1 in mouse neural tube closure

Previous studies identified Inka1 as a gene regulated by AP‐2α in the neural crest required for craniofacial morphogenesis in fish and frog. Here, we extend the analysis of Inka1 function and regulation to the mouse by generating a LacZ knock‐in allele. Inka1‐LacZ allele expression occurs in the cephalic mesenchyme, heart, and paraxial mesoderm prior to E8.5. Subsequently, expression is observed in the migratory neural crest cells and their derivatives. Consistent with expression of Inka1 in tissues of the developing head during neurulation, a low percentage of Inka1^?/? mice show exencephaly while the remainder are viable and fertile. Further studies indicate that AP‐2α is not required for Inka1 expression in the mouse, and suggest that there is no significant genetic interaction between these two factors during embryogenesis. Together, these data demonstrate that while the expression domain of Inka1 is conserved among vertebrates, its function and regulation are not. Developmental Dynamics 239:1188–1196, 2010. © 2010 Wiley‐Liss, Inc.     

Transforming growth factor Beta2 is required for valve remodeling during heart development

Although the function of transforming growth factor beta2 (TGFβ2) in epithelial mesenchymal transition (EMT) is well studied, its role in valve remodeling remains to be fully explored. Here, we used histological, morphometric, immunohistochemical and molecular approaches and showed that significant dysregulation of major extracellular matrix (ECM) components contributed to valve remodeling defects in Tgfb2^?/? embryos. The data indicated that cushion mesenchymal cell differentiation was impaired in Tgfb2^?/? embryos. Hyaluronan and cartilage link protein‐1 (CRTL1) were increased in hyperplastic valves of Tgfb2^?/? embryos, indicating increased expansion and diversification of cushion mesenchyme into the cartilage cell lineage during heart development. Finally, Western blot and immunohistochemistry analyses indicate that the activation of SMAD2/3 was decreased in Tgfb2^?/? embryos during valve remodeling. Collectively, the data indicate that TGFβ2 promotes valve remodeling and differentiation by inducing matrix organization and suppressing cushion mesenchyme differentiation into cartilage cell lineage during heart development. Developmental Dynamics 240:2127–2141, 2011. © 2011 Wiley‐Liss, Inc.     

TRPM6 and TRPM7: Novel players in cell intercalation during vertebrate embryonic development

A common theme in organogenesis is how the final structure of organs emerge from epithelial tube structures, with the formation of the neural tube being one of the best examples. Two types of cell movements co-occur during neural tube closure involving the migration of cells toward the midline of the embryo (mediolateral intercalation or convergent extension) as well as the deep movement of cells from inside the embryo to the outside of the lateral side of the neural plate (radial intercalation). Failure of either type of cell movement will prevent neural tube closure, which can produce a range of neural tube defects (NTDs), a common congenital disease in humans. Numerous studies have identified signaling pathways that regulate mediolateral intercalation during neural tube closure. Less understood are the pathways that govern radial intercalation. Using the Xenopus laevis system, our group reported the identification of transient receptor potential (TRP) channels, TRPM6 and TRPM7, and the Mg²⁺ ion they conduct, as novel and key factors regulating both mediolateral and radial intercalation during neural tube closure. Here we broadly discuss tubulogenesis and cell intercalation from the perspective of neural tube closure and the respective roles of TRPM7 and TRPM6 in this critical embryonic process.     

PKN2 is essential for mouse embryonic development and proliferation of mouse fibroblasts

PKN2, a member of the protein kinase N (PKN) family, has been suggested by in vitro culture cell experiments to bind to Rho/Rac GTPases and contributes to cell–cell contact and cell migration. To unravel the in vivo physiological function of PKN2, we targeted the PKN2 gene. Constitutive disruption of the mouse PKN2 gene resulted in growth retardation and lethality before embryonic day (E) 10.5. PKN2^?/? embryo did not undergo axial turning and showed insufficient closure of the neural tube. Mouse embryonic fibroblasts (MEFs) derived from PKN2^?/? embryos at E9.5 failed to grow. Cre‐mediated ablation of PKN2 in PKN2^flox/flox MEFs obtained from E14.5 embryos showed impaired cell proliferation, and cell cycle analysis of these MEFs showed a decrease in S‐phase population. Our results show that PKN2 is essential for mouse embryonic development and cell‐autonomous proliferation of primary MEFs in culture. Comparison of the PKN2^?/? phenotype with the phenotypes of PKN1 and PKN3 knockout strains suggests that PKN2 has distinct nonredundant functions in vivo, despite the structural similarity and evolutionary relationship among the three isoforms.     

G protein modulation of K2P potassium channel TASK-2

TASK-2 (K_2P5.1) is a background K⁺ channel opened by extra- or intracellular alkalinisation that plays a role in renal bicarbonate handling, central chemoreception and cell volume regulation. Here, we present results that suggest that TASK-2 is also modulated by Gβγ subunits of heterotrimeric G protein. TASK-2 was strongly inhibited when GTP-γ-S was used as a replacement for intracellular GTP. No inhibition was present using GDP-β-S instead. Purified Gβγ introduced intracellularly also inhibited TASK-2 independently of whether GTP or GDP-β-S was present. The effects of GTP-γ-S and Gβγ subunits were abolished by neutralisation of TASK-2 C terminus double lysine residues K257–K258 or K296–K297. Use of membrane yeast two hybrid (MYTH) experiments and immunoprecipitation assays using tagged proteins gave evidence for a physical interaction between Gβ1 and Gβ2 subunits and TASK-2, in agreement with expression of these subunits in proximal tubule cells. Co-immunoprecipitation was impeded by mutating C terminus K257–K258 (but not K296–K297) to alanines. Gating by extra- or intracellular pH was unaltered in GTP-γ-S-insensitive TASK-2-K257A-K258A mutant. Shrinking TASK-2-expressing cells in hypertonic solution decreased the current to 36 % of its initial value. The same manoeuvre had a significantly diminished effect on TASK-2-K257A-K258A- or TASK-2-K296-K297-expressing cells, or in cells containing intracellular GDP-β-S. Our data are compatible with the concept that TASK-2 channels are modulated by Gβγ subunits of heterotrimeric G protein. We propose that this modulation is a novel way in which TASK-2 can be tuned to its physiological functions.     

Targeted deletion of Nm23/nucleoside diphosphate kinase A and B reveals their requirement for definitive erythropoiesis in the mouse embryo

The ubiquitously expressed nucleoside diphosphate kinases (Nm23/NDPK/Awd) are a large family of multifunctional enzymes implicated in nucleic acid metabolism and in normal and abnormal development. Here, we describe the generation and characterization of NDPK A‐ and B‐deficient (Nme1^?/?/Nme2^?/?) mice in which >95% of the enzyme activity is eliminated. These mice are undersized, die perinatally, and exhibit a spectrum of hematological phenotypes including severe anemia, impaired maturation of erythrocytes, and abnormal hematopoiesis in the liver and bone marrow. Flow cytometric analysis of developing Nme1^?/?/Nme2^?/? erythroid cells indicated that the major iron transport receptor molecule TfR1 is attenuated concomitant with a reduction of intracellular iron, suggesting that TfR1 is a downstream target of NDPKs and that reduced iron in Nme1^?/?/Nme2^?/? erythroblasts is inhibiting their development. We conclude that Nm23/NDPKs play critical roles in definitive erythroid development. Our novel mouse model also links erythropoiesis and nucleotide metabolism. Developmental Dynamics 238:775–787, 2009. © 2009 Wiley‐Liss, Inc.     

Interferon‐induced TRAIL‐independent cell death in DNase II−/− embryos

The chromosomal DNA of apoptotic cells and the nuclear DNA expelled from erythroid precursors is cleaved by DNase II in lysosomes after the cells or nuclei are engulfed by macrophages. DNase II^?/? embryos suffer from lethal anemia due to IFN‐β produced in the macrophages carrying undigested DNA. Here, we show that Type I IFN induced a caspase‐dependent cell death in human epithelial cells that were transformed to express a high level of IFN type I receptor. During this death process, a set of genes was strongly activated, one of which encoded TRAIL, a death ligand. A high level of TRAIL mRNA was also found in the fetal liver of the lethally anemic DNase II^?/? embryos, and a lack of IFN type I receptor in the DNase II^?/? IFN‐IR^?/? embryos blocked the expression of TRAIL mRNA. However, a null mutation in TRAIL did not rescue the lethal anemia of the DNase II^?/? embryos, indicating that TRAIL is dispensable for inducing the apoptosis of erythroid cells in DNase II^?/? embryos, and therefore, that there is a TRAIL‐independent mechanism for the IFN‐induced apoptosis.     

Novel mutations in VANGL1 in neural tube defects

Neural tube defects (NTDs) are severe congenital malformations caused by failure of the neural tube to close during neurulation. Their etiology is complex involving both environmental and genetic factors. We have recently reported three mutations in the planar cell polarity gene VANGL1 associated with NTDs. The aim of the present study was to define the role of VANGL1 genetic variants in the development of NTDs in a large cohort of various ethnic origins. We identified five novel missense variants in VANGL1, p.Ser83Leu, p.Phe153Ser, p.Arg181Gln, p.Leu202Phe and p.Ala404Ser, occurring in sporadic and familial cases of spinal dysraphisms. All five variants affect evolutionary conserved residues and are absent from all controls analyzed. This study provides further evidence supporting the role of VANGL1 as a risk factor in the development of spinal NTDs. © 2009 Wiley‐Liss, Inc.     

Contribution of VANGL2 mutations to isolated neural tube defects

Kibar Z, Salem S, Bosoi CM, Pauwels E, De Marco P, Merello E, Bassuk AG, Capra V, Gros P. Contribution of VANGL2 mutations to isolated neural tube defects. Vangl2 was identified as the gene defective in the Looptail (Lp) mouse model for neural tube defects (NTDs). This gene forms part of the planar cell polarity (PCP) pathway, also called the non‐canonical Frizzled/Dishevelled pathway, which mediates the morphogenetic process of convergent extension essential for proper gastrulation and neural tube formation in vertebrates. Genetic defects in PCP signaling have strongly been associated with NTDs in mouse models. To assess the role of VANGL2 in the complex etiology of NTDs in humans, we resequenced this gene in a large multi‐ethnic cohort of 673 familial and sporadic NTD patients, including 453 open spina bifida and 202 closed spinal NTD cases. Six novel rare missense mutations were identified in seven patients, five of which were affected with closed spinal NTDs. This suggests that VANGL2 mutations may predispose to NTDs in approximately 2.5% of closed spinal NTDs (5 in 202), at a frequency that is significantly different from that of 0.4% (2 in 453) detected in open spina bifida patients (p = 0.027). Our findings strongly implicate VANGL2 in the genetic causation of spinal NTDs in a subset of patients and provide additional evidence for a pathogenic role of PCP signaling in these malformations.     

Rare mutations in the autophagy‐regulating gene AMBRA1 contribute to human neural tube defects

Neural tube defects (NTDs) are severe congenital malformations caused by failed neural tube closure. Recently, autophagy is revealed to play a vital role in neuroepithelium development and neurulation. Autophagy and beclin 1 regulator 1 (Ambra1) is a crucial regulator of autophagy initiation, and its deficiency in mice leads to exencephaly and/or spina bifida. However, the genetic contribution of AMBRA1 to the etiology of human NTDs remains unknown. In this study, we identified five rare missense mutations of AMBRA1 in 352 NTDs cases, which were absent in 224 matched controls. Western blotting and fluorescence puncta counting for MAP1LC3A/LC3 in HEK293T cells suggested that four of the mutations (AMBRA1 p.Thr80Met, p.Leu274Phe, p.Ser743Phe, and p.Met884Val) affected autophagy initiation to various extents. Furthermore, these four mutations also displayed loss‐of‐function effects compared with wild‐type AMBRA1 when we injected messenger RNA (mRNA) to overexpress or rescue ambra1a‐morpholino oligos (MO) knockdown in zebrafish. It is intriguing that trehalose, a natural disaccharide, could rescue ambra1a‐MO knockdown in a dose‐dependent manner independently or together with AMBRA1 mRNA. Taken together, our findings suggest that rare mutations of the autophagy regulator gene AMBRA1 may contribute to the etiology of human neural tube defects, and trehalose is a promising treatment for a subset of NTDs caused by autophagy impairment.     

Differential susceptibility to lethal endotoxaemia in mice deficient in IL-1α, IL-1β or IL-1 receptor type I

Joosten LAB, van de Veerdonk F, Vonk AG, Boerman OC, Keuter M, Fantuzzi G, Verschueren I, van der Poll T, Dinarello CA, Kullberg BJ, Van der Meer JWM, Netea MG. Differential susceptibility to lethal endotoxaemia in mice deficient in IL‐1α, IL‐1β or IL‐1 receptor type I. APMIS 2010; 118: 1000–7. The role of intereukin‐1 (IL‐1) in mortality caused by endotoxaemia remains controversial. While IL‐1 receptor antagonist (IL‐1Ra) protects mice from lethal endotoxaemia, mice deficient in IL‐1β (IL‐1β^{? /?}) display normal susceptibility to lipopolysaccharide (LPS). The aim of this study was to identify the source of these discrepancies. Mice deficient in IL‐1α, IL‐1β or IL‐1R type I were injected intraperitoneally with Escherichia coli or Salmonella typhimurium LPS. Survival of the mice was examined and compared with C57/Bl6 wild‐type mice. In addition, serum cytokine concentrations were determined after LPS challenge and in vitro cytokine production by peritoneal macrophages was analysed. Clearance of radioactive IL‐1α was examined in IL‐1α^?/? and wild‐type mice. IL‐1β^?/? mice were normally susceptible to endotoxaemia and cytokine production did not differ from that in control mice. Surprisingly, LPS mortality in IL‐1α^?/? mice was significantly greater than that in control mice, accompanied by higher interferon‐γ release. These effects were mediated by a distorted homeostasis of IL‐1RI receptors, as shown by a strongly delayed clearance of IL‐1α. In contrast to the IL‐1α^?/? and IL‐1β^?/? mice, IL‐1RI^?/? mice were completely resistant to high doses of LPS. In conclusion, IL‐1RI‐mediated signals are crucial in mediating mortality occurring as a result of lethal endotoxaemia. Investigation of IL‐1‐mediated pathways in IL‐1 knock‐out mice is complicated by a distorted homeostasis of IL‐1Rs.     

High‐affinity folate receptor in cardiac neural crest migration: A gene knockdown model using siRNA

Folate supplementation reduces the incidence of congenital heart defects, but the nature of this protective mechanism remains unclear. Immunolabeling demonstrated that the neural tube and neural crest (NC) cells were rich in the high‐affinity folate receptor FOLR1and during the early stages of development FOLR1 was found principally in these cells. Suppression of Folr1 expression in the nascent cardiac NC by site‐directed short‐interfering RNA (siRNA) altered cardiac NC cell mitosis and subsequent migration patterns leading to abnormal development of the pharyngeal arch arteries (PAA) and outflow tract. qPCR analysis demonstrated that the siRNA treatment significantly reduced Folr1 24 hr after treatment. These treatments also significantly reduced mitosis in the neural tube, but adjacent, nontreated areas were unaffected. In summary, a brief reduction in the expression of Folr1 during a critical stage of NC development had long‐term consequences for the development of the PAA and outflow tract. Developmental Dynamics 239:1136–1144, 2010. © 2010 Wiley‐Liss, Inc.