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.     

Bmp2 is required for cephalic neural tube closure in the mouse

BMPs have been shown to play a role in neural tube development particularly as dorsalizing factors. To explore the possibility that BMP2 could play a role in the developing neural tube (NT) beyond the lethality of Bmp2 null embryos, we created Bmp2 chimeras from Bmp2 null ES cells and WT blastocysts. Analysis of Bmp2 chimeras reveals NT defects at day 9.5 (E9.5). We found that exclusion of Bmp2 null ES cells from the dorsal NT did not always prevent defects. For further comparison, we used a Bmp2 mutant line in a mixed background. Phenotypes observed were similar to chimeras including open NT defects, postneurulation defects, and abnormal neural ectoderm in heterozygous and homozygous null embryos demonstrating a pattern of dose‐dependent signaling. Our data exposes BMP2 as a unique player in the developing NT for dorsal patterning and identity, and normal cephalic neural tube closure in a dose‐dependent manner. Developmental Dynamics 238:110–122, 2009. Published 2008 Wiley‐Liss, Inc.     

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.     

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 tube defects and impaired neural progenitor cell proliferation in Gβ1‐deficient mice

Heterotrimeric G proteins are well known for their roles in signal transduction downstream of G protein–coupled receptors (GPCRs), and both Gα subunits and tightly associated Gβγ subunits regulate downstream effector molecules. Compared to Gα subunits, the physiological roles of individual Gβ and Gγ subunits are poorly understood. In this study, we generated mice deficient in the Gβ1 gene and found that Gβ1 is required for neural tube closure, neural progenitor cell proliferation, and neonatal development. About 40% Gβ1^?/? embryos developed neural tube defects (NTDs) and abnormal actin organization was observed in the basal side of neuroepithelium. In addition, Gβ1^?/? embryos without NTDs showed microencephaly and died within 2 days after birth. GPCR agonist‐induced ERK phosphorylation, cell proliferation, and cell spreading, which were all found to be regulated by Gαi and Gβγ signaling, were abnormal in Gβ1^?/? neural progenitor cells. These data indicate that Gβ1 is required for normal embryonic neurogenesis. Developmental Dynamics 239:1089–1101, 2010. © 2010 Wiley‐Liss, Inc.     

Development of the nuclei and cell migration in the medulla oblongata

Summary We have tried to obtain new insight into the development of the medulla oblongata by using the quail-to-chick chimera system. Five types of isotopic and isochrome grafts were carried out, between quail and chick embryos, at the 10- to 12-somite stage: exchanges of (I) the entire myelencephalon, (II) the dorsal half of the myelencephalon, (III) the ventral half of the myelencephalon, (IV) the right half of the myelencephalon and (V) the dorsal quarter of the myelencephalon. Before analyzing the chimeric embryos, we studied the ontogeny of the various nuclei in the medulla oblongata of normal birds. The first appearance of nuclei in quail embryos preceded in many cases that of their chick counterpart by 12 to 24 h. The adult pattern of the nuclei was established by E8 in quail and E9 in chick. Similarly, during early development of chimeras, the migration of quail cells began earlier than that of chick cells. This shows that the species specific temporal sequence of proliferation and migration is not significantly altered by transplantation into the host. The possibility of grafting selectively the ventral or dorsal half of the neural tube allowed us to distinguish the fate of the cells belonging respectively to the alar and the basal plate. The nuclei with a total or partial motor function, such as the nucleus nervi abducentis, the nucleus nervi facialis, the nucleus nervi glossopharyngei and the nucleus motorius dorsalis nervi vagi, have either an exclusive or predominant origin from the basal plate. In contrast, the nuclei with essentially or exclusively sensory components (i.e., nucleus angularis, nucleus laminaris, nucleus magnocellularis) arise from the alar plate. The reticular formation such as the nucleus reticularis gigantocellularis and the nucleus reticularis subtrigeminalis was strikingly mixed, with both alar and basal plate origin of neurons. Active dorsoventral migrations of cells originating migrations from the dorsal neural tube, the rhombic lip, contribute the ventral nuclei (i.e., nuclei pontis medialis, lateralis and olivaris inferior), whose functions are essentially associative. This study shows different types of cell migration. Dorsoventral and ventrodorsal movements are essentially active from E5 to E8. In the medulla oblongata, the dorsoventral stream is highly predominant. From E8 to E9, cells belonging to the marginal stream cross the midline laterally in both directions. Beyond E12, longitudinal migrations occur ventrally in both rostrocaudal and caudorostral directions. The immunohistochemical analyses carried out on chimeras generated in experiment V revealed the existence of fibers in marginal zones prior to the onset of the migration of cell bodies. These observations support the suggestion that such tangential fibers serve as guidance substrates for the subpial migrations of cells in the medulla oblongata.     

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.     

Wnt signaling plays an essential role in neuronal specification of the dorsal spinal cord

In the developing spinal cord, signals from the roof plate are required for the development of three classes of dorsal interneuron: D1, D2, and D3, listed from dorsal to ventral. Here, we demonstrate that absence of Wnt1 and Wnt3a, normally expressed in the roof plate, leads to diminished development of D1 and D2 neurons and a compensatory increase in D3 neuron populations. This occurs without significantly altered expression of BMP and related genes in the roof plate. Moreover, Wnt3a protein induces expression of D1 and D2 markers in the isolated medial region of the chick neural plate, and Noggin does not interfere with this induction. Thus, Wnt signaling plays a critical role in the specification of cell types for dorsal interneurons.     

Lack of lipid phosphate phosphatase-3 in embryonic stem cells compromises neuronal differentiation and neurite outgrowth

Background: Bioactive lipids such as lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P) have been recently described as important regulators of pluripotency and differentiation of embryonic stem (ES) cells and neural progenitors. Due to the early lethality of LPP3, an enzyme that regulates the levels and biological activities of the aforementioned lipids, it has been difficult to assess its participation in early neural differentiation and neuritogenesis. Results: We find that Ppap2b^?/? (Lpp3^?/?) ES cells differentiated in vitro into spinal neurons show a considerable reduction in the amount of neural precursors and young neurons formed. In addition, differentiated Lpp3^?/? neurons exhibit impaired neurite outgrowth. Surprisingly, when Lpp3^?/? ES cells were differentiated, an unexpected appearance of smooth muscle actin‐positive cells was observed, an event that was partially dependent upon phosphorylated sphingosines. Conclusions: Our data show that LPP3 plays a fundamental role during spinal neuron differentiation from ES and that it also participates in regulating neurite and axon outgrowth. Developmental Dynamics 241:953–964, 2012. © 2012 Wiley Periodicals, Inc.     

A direct role for Wnt8 in ventrolateral mesoderm patterning

Vertebrate dorsoventral patterning requires both Wnt8 and BMP signaling. Because of their multiple interactions, discerning roles attributable specifically to Wnt8 independent of BMP has been a challenge. For example, Wnt8 represses the dorsal organizer that negatively regulates ventral BMP signals, thus Wnt8 loss‐of‐function phenotypes may reflect the combined effects of reduced Wnt8 and BMP signaling. We have taken a loss‐of‐function approach in the zebrafish to generate embryos lacking expression of both Wnt8 and the BMP antagonist Chordin. wnt8;chordin loss‐of‐function embryos show rescued BMP signaling, thereby allowing us to identify Wnt8‐specific requirements. Our analysis shows that Wnt8 is uniquely required to repress prechordal plate specification but not notochord, and that Wnt8 signaling is not essential for specification of tailbud progenitors but is required for normal expansion of posterior mesoderm cell populations. Thus, Wnt8 and BMP signaling have independent roles during vertebrate ventrolateral mesoderm development that can be identified through loss‐of‐function analysis. Developmental Dynamics 239:2828–2836, 2010. © 2010 Wiley‐Liss, Inc.     

Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite

Embryonic patterning in vertebrates is dependent upon the balance of inductive signals and their specific antagonists. We show that Noggin, which encodes a bone morphogenetic protein (BMP) antagonist expressed in the node, notochord, and dorsal somite, is required for normal mouse development. Although Noggin has been implicated in neural induction, examination of null mutants in the mouse indicates that Noggin is not essential for this process. However, Noggin is required for subsequent growth and patterning of the neural tube. Early BMP-dependent dorsal cell fates, the roof plate and neural crest, form in the absence of Noggin. However, there is a progressive loss of early, Sonic hedgehog (Shh)-dependent ventral cell fates despite the normal expression of Shh in the notochord. Further, somite differentiation is deficient in both muscle and sclerotomal precursors. Addition of BMP2 or BMP4 to paraxial mesoderm explants blocks Shh-mediated induction of Pax-1, a sclerotomal marker, whereas addition of Noggin is sufficient to induce Pax-1. Noggin and Shh induce Pax-1 synergistically. Use of protein kinase A stimulators blocks Shh-mediated induction of Pax-1, but not induction by Noggin, suggesting that induction is mediated by different pathways. Together these data demonstrate that inhibition of BMP signaling by axially secreted Noggin is an important requirement for normal patterning of the vertebrate neural tube and somite.     

Regulation of Sox9 by Sonic Hedgehog (Shh) is essential for patterning and formation of tracheal cartilage

We report that Sonic Hedgehog (Shh) regulates both formation and patterning of tracheal cartilage by controlling the expression pattern and level of the chondrogenic gene, Sox9. In Shh^?/? tracheo‐esophageal tubes, Sox9 expression is transient and not restricted ventrally to the site of chondrogenesis, and is absent at the time of chondrogenesis, resulting in the failure of tracheal cartilage formation. Inhibition of Hedgehog signalling with cyclopamine in tracheal cultures prevents tracheal cartilage formation, while treatment of Shh^?/? tracheal explant with exogenous Shh peptide rescues cartilage formation. Both exogenous Bmp4 and Noggin rescue cartilage phenotype in Shh^?/? tracheal culture, while promoting excessive cartilage development in wild‐type trachea through induction of Sox9 expression. The ventral and segmented expression of Sox9 in tracheal primordia under Shh modulated by Bmp4 and Noggin thus determine where and when tracheal cartilage develops. These results indicate that Shh signalling is a critical determinant in tracheal cartilage development. Developmental Dynamics 239:514–526, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Impaired T‐cell development in the absence of Vav1 and Itk

Vav1 and the Tec family kinase Itk act in similar T‐cell activation pathways. Both molecules interact with members of the Cbl family of E3 ubiquitin ligases, and signaling defects in Vav1^?/? T cells are rescued upon deletion of Cbl‐b. In this study we investigate the relation between Itk and Cbl‐b or Vav1 by generating Itk/Cbl‐b and Itk/Vav1 double‐deficient mice. Deletion of Cbl‐b in Itk^?/? CD4⁺ T cells restored proliferation and partially IL‐2 production, and also led to a variable rescue of IL‐4 production. Thus, Itk and Vav1 act mechanistically similarly in peripheral T cells, since the defects in Itk^?/? T cells, as in Vav1^?/? T cells, are rescued if cells are released from the negative regulation mediated by Cbl‐b. In addition, only few peripheral CD4⁺ and CD8⁺ T cells were present in Vav1^?/?Itk^?/? mice due to severely impaired thymocyte differentiation. Vav1^?/?Itk^?/? thymocyte numbers were strongly reduced compared with WT, Itk^?/? or Vav1^?/? mice, and double‐positive thymocytes displayed increased cell death and impaired positive selection. Therefore, our data also reveal that the combined activity of Vav1 and Itk is required for proper T‐cell development and the generation of the peripheral T‐cell pool.     

Sox10 overexpression induces neural crest-like cells from all dorsoventral levels of the neural tube but inhibits differentiation.

SoxE genes (Sox8, Sox9, and Sox10) are early response genes to neural crest induction. Although the early role of Sox9 has been examined in chick and frog, later roles in neural crest migration and differentiation remain largely unexplored. We first examined which SoxE genes were expressed in trunk neural crest cells and then investigated their function using in ovo electroporation. The results of this analysis reveal that Sox10 is present in migrating neural crest cells, whereas other SoxE genes are only expressed transiently after induction. Ectopic expression of Sox10 in the neural tube at trunk level induced expression of HNK-1 in neuroepithelial cells followed by extensive emigration from all levels of the dorsoventral neuraxis, including the floor plate. Sox10-expressing cells failed to express neuronal, Schwann, or melanocyte markers up to 6 days posttransfection (E8), suggesting these cells were maintained in an undifferentiated state. Overexpression of Sox8 or Sox9 had similar but not identical effects on neuroepithelial cells. These results show that high levels of Sox10, Sox9, or Sox8 expression in the neural tube are capable of inducing a migratory neural crest-like phenotype even in the absence of dorsal signals and can maintain these cells in an undifferentiated state.     

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.     

Gadd45b and Gadd45g are important for anti‐tumor immune responses

An effective Th1 type cell‐mediated immune response against cancer cells is critical in limiting cancer progression. Gadd45b, a signaling molecule highly up‐regulated during Th1 type responses, is studied for its role in limiting tumor growth. Mouse B16 melanoma cells implanted into Gadd45b^?/? mice grew faster than those in WT or Gadd45b^+/? littermate controls. The defect of Gadd45b^?/? mice in tumor immunosurveillance was attributed to the reduced expression of IFN‐γ, granzyme B, and CCR5 in Gadd45b^?/? CD8⁺ T cells at the tumor site. Activation of p38 MAP kinase, but not ERK or JNK, by either TCR‐stimuli or IL‐12 and IL‐18 is diminished in Gadd45b^?/? CD8⁺ T cells, resulting in reduced production of IFN‐γ. In addition, mRNA of T‐bet and Eomes were reduced in Gadd45b^?/? CD8⁺ T cells, supporting a critical role of Gadd45b in shaping the Th1 fate. More importantly, the tumor vaccination, which is effective in WT mice, failed in Gadd45b/Gadd45g doubly deficient mice. Collectively, these data demonstrate that members of the Gadd45 gene family are important for anti‐tumor immune responses.     

Bone morphogenetic protein signaling is required in the dorsal neural folds before neurulation for the induction of spinal neural crest cells and dorsal neurons

Bone Morphogenetic Protein (BMP) activity has been implicated as a key regulator of multiple aspects of dorsal neural tube development. BMP signaling in the dorsal‐most neuroepithelial cells presumably plays a critical role. We use tissue‐specific gene ablation to probe the roles of BMPR1A, the type 1 BMP receptor that is seemingly the best candidate to mediate the activities of BMPs on early dorsal neural development. We use two different Cre lines expressed in the dorsal neural folds, one prior to spinal neurulation and one shortly afterward, together with a Bmpr1a conditional null mutation. Our findings indicate that BMPR1A signaling in the dorsal neural folds is important for hindbrain neural tube closure, but suggest it is dispensable for spinal neurulation. Our results also demonstrate a requirement for BMP signaling in patterning of dorsal neural tube cell fate and in neural crest cell formation, and imply a critical period shortly before neural tube closure. Developmental Dynamics 240:755–765, 2011. © 2011 Wiley‐Liss, Inc.