Dissecting the roles of ROCK isoforms in stress-induced cell detachment

The homologous Rho kinases, ROCK1 and ROCK2, are involved in stress fiber assembly and cell adhesion and are assumed to be functionally redundant. Using mouse embryonic fibroblasts (MEFs) derived from ROCK1^−/− and ROCK2^−/− mice, we have recently reported that they play different roles in regulating doxorubicin-induced stress fiber disassembly and cell detachment: ROCK1 is involved in destabilizing the actin cytoskeleton and cell detachment, whereas ROCK2 is required for stabilizing the actin cytoskeleton and cell adhesion. Here, we present additional insights into the roles of ROCK1 and ROCK2 in regulating stress-induced impairment of cell-matrix and cell-cell adhesion. In response to doxorubicin, ROCK1^−/− MEFs showed significant preservation of both focal adhesions and adherens junctions, while ROCK2^−/− MEFs exhibited impaired focal adhesions but preserved adherens junctions compared with the wild-type MEFs. Additionally, inhibition of focal adhesion or adherens junction formations by chemical inhibitors abolished the anti-detachment effects of ROCK1 deletion. Finally, ROCK1^−/− MEFs, but not ROCK2^−/− MEFs, also exhibited preserved central stress fibers and reduced cell detachment in response to serum starvation. These results add new insights into a novel mechanism underlying the anti-detachment effects of ROCK1 deletion mediated by reduced peripheral actomyosin contraction and increased actin stabilization to promote cell-cell and cell-matrix adhesion. Our studies further support the differential roles of ROCK isoforms in regulating stress-induced loss of central stress fibers and focal adhesions as well as cell detachment.     

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1^−/− and ROCK2^−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1^−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2^−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.     

Altered bone development and an increase in FGF-23 expression in Enpp1(-/-) mice

Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is required for the conversion of extracellular ATP into inorganic pyrophosphate (PP_i), a recognised inhibitor of hydroxyapatite (HA) crystal formation. A detailed phenotypic assessment of a mouse model lacking NPP1 (Enpp1^−/−) was completed to determine the role of NPP1 in skeletal and soft tissue mineralization in juvenile and adult mice. Histopathological assessment of Enpp1^−/− mice at 22 weeks of age revealed calcification in the aorta and kidney and ectopic cartilage formation in the joints and spine. Radiographic assessment of the hind-limb showed hyper-mineralization in the talocrural joint and hypo-mineralization in the femur and tibia. MicroCT analysis of the tibia and femur disclosed altered trabecular architecture and bone geometry at 6 and 22 weeks of age in Enpp1^−/− mice. Trabecular number, trabecular bone volume, structure model index, trabecular and cortical thickness were all significantly reduced in tibiae and femurs from Enpp1^−/− mice (P<0.05). Bone stiffness as determined by 3-point bending was significantly reduced in Enpp1^−/− tibiae and femurs from 22-week-old mice (P<0.05). Circulating phosphate and calcium levels were reduced (P<0.05) in the Enpp1^−/− null mice. Plasma levels of osteocalcin were significantly decreased at 6 weeks of age (P<0.05) in Enpp1^−/− mice, with no differences noted at 22 weeks of age. Plasma levels of CTx (Ratlaps™) and the phosphaturic hormone FGF-23 were significantly increased in the Enpp1^−/− mice at 22 weeks of age (P<0.05). Fgf-23 messenger RNA expression in cavarial osteoblasts was increased 12-fold in Enpp1^−/− mice compared to controls. These results indicate that Enpp1^−/− mice are characterized by severe disruption to the architecture and mineralization of long-bones, dysregulation of calcium/phosphate homeostasis and changes in Fgf-23 expression. We conclude that NPP1 is essential for normal bone development and control of physiological bone mineralization.     

Barx1-mediated inhibition of Wnt signaling in the mouse thoracic foregut controls tracheo-esophageal septation and epithelial differentiation

Mesenchymal cells underlying the definitive endoderm in vertebrate animals play a vital role in digestive and respiratory organogenesis. Although several signaling pathways are implicated in foregut patterning and morphogenesis, and despite the clinical importance of congenital tracheal and esophageal malformations in humans, understanding of molecular mechanisms that allow a single tube to separate correctly into the trachea and esophagus is incomplete. The homoebox gene Barx1 is highly expressed in prospective stomach mesenchyme and required to specify this organ. We observed lower Barx1 expression extending contiguously from the proximal stomach domain, along the dorsal anterior foregut mesenchyme and in mesenchymal cells between the nascent esophagus and trachea. This expression pattern exactly mirrors the decline in Wnt signaling activity in late development of the adjacent dorsal foregut endoderm and medial mainstem bronchi. The hypopharynx in Barx1^−/− mouse embryos is abnormally elongated and the point of esophago-tracheal separation shows marked caudal displacement, resulting in a common foregut tube that is similar to human congenital tracheo-esophageal fistula and explains neonatal lethality. Moreover, the Barx1^−/− esophagus displays molecular and cytologic features of respiratory endoderm, phenocopying abnormalities observed in mouse embryos with activated ß-catenin. The zone of canonical Wnt signaling is abnormally prolonged and expanded in the proximal Barx1^−/− foregut. Thus, as in the developing stomach, but distinct from the spleen, Barx1 control of thoracic foregut specification and tracheo-esophageal septation is tightly associated with down-regulation of adjacent Wnt pathway activity.     

Visceral Endoderm Expression of Yin-Yang1 (YY1) Is Required for VEGFA Maintenance and Yolk Sac Development

Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implantation stage. To assess the post-gastrulation role of YY1, a conditional knock-out (cKO) strategy was used to delete YY1 from the visceral endoderm of the yolk sac and the definitive endoderm of the embryo. cKO embryos display profound yolk sac defects at 9.5 days post coitum (dpc), including disrupted angiogenesis in mesoderm derivatives and altered epithelial characteristics in the visceral endoderm. Significant changes in both cell death and proliferation were confined to the YY1-expressing yolk sac mesoderm indicating that loss of YY1 in the visceral endoderm causes defects in the adjacent yolk sac mesoderm. Production of Vascular Endothelial Growth Factor A (VEGFA) by the visceral endoderm is essential for normal growth and development of the yolk sac vasculature. Reduced levels of VEGFA are observed in the cKO yolk sac, suggesting a cause for the angiogenesis defects. Ex vivo culture with exogenous VEGF not only rescued angiogenesis and apoptosis in the cKO yolk sac mesoderm, but also restored the epithelial defects observed in the cKO visceral endoderm. Intriguingly, blocking the activity of the mesoderm-localized VEGF receptor, FLK1, recapitulates both the mesoderm and visceral endoderm defects observed in the cKO yolk sac. Taken together, these results demonstrate that YY1 is responsible for maintaining VEGF in the developing visceral endoderm and that a VEGF-responsive paracrine signal, originating in the yolk sac mesoderm, is required to promote normal visceral endoderm development.     

Stage-specific embryonic antigen 3 as a marker of visceral extraembryonic endoderm

The distribution of the stage-specific embryonic antigen SSEA-3 was studied immunohistochemically on postimplantation mouse embryos. This carbohydrate antigen, identified as an epitope of a globo-series ganglioside isolated from human teratocarcinoma cells (Kannagi et al., 1983, J. Biol. Chem.258, 8934–8942) was originally detected on the zygote and mouse early cleavage-stage embryos. It disappears on the early blastocyst and reappears on the primitive endoderm of the implanting blastocyst (Shevinsky et al., 1982, Cell30, 697–705). We now show in the early egg cylinder (on the sixth day of pregnancy) SSEA-3 is present in the entire visceral endoderm but not in any other part of the conceptus. From Day 7 of pregnancy onward, SSEA-3 is restricted to the extraembryonic visceral endoderm and the visceral yolk sac cells. Therefore, SSEA-3 is a useful marker for this endodermal cell lineage in midgestational mouse embryos.     

Transthyretin mouse transgenes direct RFP expression or Cre‐mediated recombination throughout the visceral endoderm

Transthyretin (Ttr) is a thyroid hormone transport protein secreted by cells of the visceral yolk sac and fetal liver in developing embryos, and by hepatocytes and the choroid plexus epithelium of the brain in adult mice. Spatiotemporal localization of Ttr mRNA during embryogenesis suggested that Ttr regulatory elements might drive transgene expression throughout the visceral endoderm of early mouse embryos. We use Ttr cis‐regulatory elements to generate Ttr::RFP and Ttr::Cre strains of mice, driving red fluorescent protein (RFP) and a nuclear‐localized Cre recombinase, respectively. Visualization of RFP fluorescence in Ttr::RFP transgenics confirms reporter localization throughout the visceral endoderm in early embryos and in the visceral yolk sac and fetal liver of later stage embryos. Using both GFP‐based and LacZ‐based Cre reporter strains, we demonstrate that in Ttr::Cre transgenics, Cre‐mediated recombination occurs throughout the visceral endoderm. The Ttr::Cre strain can therefore be used as a tool for genetic modifications within the visceral endoderm lineage. genesis 47:447–455, 2009. © 2009 Wiley‐Liss, Inc.     

The effects of high-sucrose diets and of maternal diabetes on the ultrastructure of the visceral yolk sac endoderm in rat embryos developing in vivo and in vitro

The ultrastructure of the visceral yolk sac endoderm of in vivo developing 9- to 13-day-old embryos from 2 diabetic rat models (streptozotocin diabetes and Cohen--genetically determined--diabetes) and from nondiabetic rats fed high sucrose diets have been studied. This was compared to yolk sacs from 9.5-day-old embryos cultured for 48 h in sera from diabetic and nondiabetic rats fed a high-sucrose diet. Light-microscopic, TEM and SEM studies showed that the pathological cellular changes in the visceral yolk sac endoderm from diabetic rats were first observed on day 9 and were most severe among 11-day-old embryos. In vitro culture of control rat embryos in serum from experimental animals induced a reduction in the number of microvilli, of vacuolar intracellular inclusions and an increase in the number of degenerated endodermal cells. SEM studies showed that in addition to disappearance of microvilli, the majority of cells were collapsed and had degenerated cell membranes. Culture of embryos from diabetic animals in control serum only slightly reversed the pathological changes in the visceral yolk sac endoderm. A good correlation exists between the rate of embryonic malformations in diabetic rats and an index of endodermal-cell damage in the visceral yolk sac.     

Cell lineage analysis of the primitive and visceral endoderm of mouse embryos cultured in vitro

Cell lineages of the primitive endoderm and the visceral endoderm of mouse embryos were examined by culturing whole embryos in vitro. The primitive endoderm and visceral endoderm cells could be labelled by incubation of embryos in a medium containing horse radish peroxidase (HRP). HRP localization was chased throughout the culture period. The results show that the visceral endoderm derives from the primitive endoderm, and the visceral endoderm forms only the extra-embryonic endoderm (yolk sac endoderm) of the conceptus. The definitive endoderm which is probably derived from the head process, newly appears on the ventral surface of the embryo.     

Autotaxin in embryonic development

Autotaxin (ATX) is a secreted lysophospholipase D that generates the multifunctional lipid mediator lysophosphatidic acid (LPA). LPA signals through six distinct G protein-coupled receptors, acting alone or in concert to activate multiple effector pathways. The ATX–LPA signaling axis is implicated in a remarkably wide variety of physiological and pathological processes and plays a vital role in embryonic development. Disruption of the ATX-encoding gene (Enpp2) in mice results in intrauterine death due to vascular defects in the extra-embryonic yolk sac and embryo proper. In addition, Enpp2 (−/−) embryos show impaired neural development. The observed angiogenic defects are attributable, at least in part, to loss of LPA signaling through the Gα_12/13-linked RhoA-ROCK-actin remodeling pathway. Studies in zebrafish also have uncovered a dual role for ATX in both vascular and neural development; furthermore, they point to a key role for ATX–LPA signaling in the regulation of left–right asymmetry. Here we discuss our present understanding of the role of ATX–LPA signaling in vertebrate development. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Expression of Actin-interacting Protein 1 Suppresses Impaired Chemotaxis of Dictyostelium Cells Lacking the Na+-H+ Exchanger NHE1

Increased intracellular pH is an evolutionarily conserved signal necessary for directed cell migration. We reported previously that in Dictyostelium cells lacking H⁺ efflux by a Na⁺-H⁺ exchanger (NHE; Ddnhe1⁻), chemotaxis is impaired and the assembly of filamentous actin (F-actin) is attenuated. We now describe a modifier screen that reveals the C-terminal fragment of actin-interacting protein 1 (Aip1) enhances the chemotaxis defect of Ddnhe1⁻ cells but has no effect in wild-type Ax2 cells. However, expression of full-length Aip1 mostly suppresses chemotaxis defects of Ddnhe1⁻ cells and restores F-actin assembly. Aip1 functions to promote cofilin-dependent actin remodeling, and we found that although full-length Aip1 binds cofilin and F-actin, the C-terminal fragment binds cofilin but not F-actin. Because pH-dependent cofilin activity is attenuated in mammalian cells lacking H⁺ efflux by NHE1, our current data suggest that full-length Aip1 facilitates F-actin assembly when cofilin activity is limited. We predict the C-terminus of Aip1 enhances defective chemotaxis of Ddnhe1⁻ cells by sequestering the limited amount of active cofilin without promoting F-actin assembly. Our findings indicate a cooperative role of Aip1 and cofilin in pH-dependent cell migration, and they suggest defective chemotaxis in Ddnhe1⁻ cells is determined primarily by loss of cofilin-dependent actin dynamics.     

The notch ligand delta-like 4 regulates multiple stages of early hemato-vascular development

Background

In mouse embryos, homozygous or heterozygous deletions of the gene encoding the Notch ligand Dll4 result in early embryonic death due to major defects in endothelial remodeling in the yolk sac and embryo. Considering the close developmental relationship between endothelial and hematopoietic cell lineages, which share a common mesoderm-derived precursor, the hemangioblast, and many key regulatory molecules, we investigated whether Dll4 is also involved in the regulation of early embryonic hematopoiesis.

Methodology/Principal Findings

Using Embryoid Bodies (EBs) derived from embryonic stem cells harboring hetero- or homozygous Dll4 deletions, we observed that EBs from both genotypes exhibit an abnormal endothelial remodeling in the vascular sprouts that arise late during EB differentiation, indicating that this in vitro system recapitulates the angiogenic phenotype of Dll4 mutant embryos. However, analysis of EB development at early time points revealed that the absence of Dll4 delays the emergence of mesoderm and severely reduces the number of blast-colony forming cells (BL-CFCs), the in vitro counterpart of the hemangioblast, and of endothelial cells. Analysis of colony forming units (CFU) in EBs and yolk sacs from Dll4^+/− and Dll4^−/− embryos, showed that primitive erythropoiesis is specifically affected by Dll4 insufficiency. In Dll4 mutant EBs, smooth muscle cells (SMCs) were seemingly unaffected and cardiomyocyte differentiation was increased, indicating that SMC specification is Dll4-independent while a normal dose of this Notch ligand is essential for the quantitative regulation of cardiomyogenesis.

Conclusions/Significance

This study highlights a previously unnoticed role for Dll4 in the quantitative regulation of early hemato-vascular precursors, further indicating that it is also involved on the timely emergence of mesoderm in early embryogenesis.     

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH ^−/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH ^−/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH ^−/− iPS cell lines, we aggregated FAH ^−/−-iPS cells with tetraploid embryos and obtained entirely FAH ^−/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH ^−/− mice. Then, we transduced FAH cDNA into the FAH ^−/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.     

Cell fate, morphogenetic movement and population kinetics of embryonic endoderm at the time of germ layer formation in the mouse

The fate of the embryonic endoderm (generally called visceral embryonic endoderm) of prestreak and early primitive streak stages of the mouse embryo was studied in vitro by microinjecting horseradish peroxidase into single axial endoderm cells of 6.7-day-old embryos and tracing the labelled descendants either through gastrulation (1 day of culture) or to early somite stages (2 days of culture). Descendants of endoderm cells from the anterior half of the axis were found at the extreme cranial end of the embryo after 1 day and in the visceral yolk sac endoderm after 2 days, i.e. they were displaced anteriorly and anterolaterally. Descendants of cells originating over and near the anterior end of the early primitive streak, i.e. posterior to the distal tip of the egg cylinder, were found after 1 day over the entire embryonic axis and after 2 days in the embryonic endoderm at the anterior intestinal portal, in the foregut, along the trunk and postnodally, as well as anteriorly and posteriorly in the visceral yolk sac. Endoderm covering the posterior half of the early primitive streak contributed to postnodal endoderm after 1 day (at the late streak stage) and mainly to posterior visceral yolk sac endoderm after 2 days. Clonal descendants of axial endoderm were located after 2 days either over the embryo or in the yolk sac; the few exceptions spanned the caudal end of the embryo and the posterior yolk sac. The clonal analysis also showed that the endoderm layer along the posterior half of the axis of prestreak- and early-streak-stage embryos is heterogeneous in its germ layer fate. Whereas the germ layer location of descendants from anterior sites did not differ after 1 day from that expected from the initial controls (approx. 90% exclusively in endoderm), only 62% of the successfully injected posterior sites resulted in labelled cells exclusively in endoderm; the remainder contributed partially or entirely to ectoderm and mesoderm. This loss from the endoderm layer was compensated by posterior-derived cells that remained in endoderm having more surviving descendants (8.4 h population doubling time) than did anterior-derived cells (10.5 h population doubling time). There was no indication of cell death at the prestreak and early streak stages; at least 93% of the cells were proliferating and more than half of the total axial population were in, or had completed, a third cell cycle after 22 h culture.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bmpr1a is required for proper migration of the AVE through regulation of Dkk1 expression in the pre-streak mouse embryo

Here, we report a novel mechanism regulating migration of the anterior visceral endoderm (AVE) by BMP signaling through BMPRIA. In Bmpr1a-deficient (Bmpr-null) embryos, the AVE does not migrate at all. In embryos with an epiblast-specific deletion of Bmpr1a (Bmpr1a^null/flox; Sox2Cre embryos), the AVE cells migrate randomly from the distal end of embryos, resulting in an expansion of the AVE. Dkk1, which is normally expressed in the anterior proximal visceral endoderm (PxVE), is downregulated in Bmpr-null embryos, whereas it is circumferentially expressed in Bmpr1a^null/flox; Sox2Cre embryos at E5.75-6.5. These results demonstrate an association of the position of Dkk1 expressing cells with direction of the migration of AVE. In Bmpr1a^null/flox; Sox2Cre embryos, a drastic decrease of WNT signaling is observed at E6.0. Addition of WNT3A to the culture of Bmpr1a^null/flox; Sox2Cre embryos at E5.5 restores expression patterns of Dkk1 and Cer1. These data indicate that BMP signaling in the epiblast induces Wnt3 and Wnt3a expression to maintain WNT signaling in the VE, resulting in downregulation of Dkk1 to establish the anterior expression domain. Thus, our results suggest that BMP signaling regulates the expression patterns of Dkk1 for anterior migration of the AVE.