Ephrin-B1 forward and reverse signaling are required during mouse development

Eph receptors and ephrin ligands are key players in many developmental processes including embryo patterning, angiogenesis, and axon guidance. Eph/ephrin interactions lead to the generation of a bidirectional signal, in which both the Eph receptors and the ephrins activate downstream signaling cascades simultaneously. To understand the role of ephrin-B1 and the importance of ephrin-B1-induced reverse signaling during embryonic development, we have generated mouse lines carrying mutations in the efnb1 gene. Complete ablation of ephrin-B1 resulted in perinatal lethality associated with a range of phenotypes, including defects in neural crest cell (NCC)-derived tissues, incomplete body wall closure, and abnormal skeletal patterning. Conditional deletion of ephrin-B1 demonstrated that ephrin-B1 acts autonomously in NCCs, and controls their migration. Last, a mutation in the PDZ binding domain indicated that ephrin-B1-induced reverse signaling is required in NCCs. Our results demonstrate that ephrin-B1 acts both as a ligand and as a receptor in a tissue-specific manner during embryogenesis.     

Requirements for Jag1-Rbpj mediated Notch signaling during early mouse lens development

Background: During vertebrate lens development, the lens placode in the embryonic ectoderm invaginates into a lens vesicle, which then separates from the surface epithelium, followed by two waves of fiber cell differentiation. In the mouse, multiple labs have shown that Jag1‐Notch signaling is critically required during the second wave of lens fiber cell formation. However, Notch signaling appears to play no obvious role during lens induction or morphogenesis, although multiple pathway genes are expressed at these earlier stages. Results: Here, we explored functions for Notch signaling specifically during early lens development, by using the early‐acting AP2α‐Cre driver to delete Jag1 or Rbpj. We found that Jag1 and Rbpj are not required during lens induction, but are necessary for proper lens vesicle separation from the surface ectoderm. Conclusions: We conclude that precise levels of Notch signaling are essential during lens vesicle morphogenesis. In addition, AP2α‐Cre‐mediated deletion of Rbpj resulted in embryos with cardiac outflow tract and liver deformities, and perinatal lethality. Developmental Dynamics 241:493–504, 2012. © 2012 Wiley Periodicals, Inc.     

Feedback control of mammalian Hedgehog signaling by the Hedgehog-binding protein,Hip1, modulates Fgf signaling during branching morphogenesis of the lung

Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development. A key issue is how negative regulation of Hh signaling might contribute to generating differential responses over tens of cell diameters. In cells that respond to Hh, two proteins that are up-regulated are Patched1 (Ptch1), the Hh receptor, a general target in both invertebrate and vertebrate organisms, and Hip1, a Hh-binding protein that is vertebrate specific. To address the developmental role of Hip1 in the context of Hh signaling, we generated Hip1 mutants in the mouse. Loss of Hip1 function results in specific defects in two Hh target issues, the lung, a target of Sonic hedgehog (Shh) signaling, and the endochondral skeleton, a target of Indian hedgehog (Ihh) signaling. Hh signaling was up-regulated in Hip1 mutants, substantiating Hip1's general role in negatively regulating Hh signaling. Our studies focused on Hip1 in the lung. Here, a dynamic interaction between Hh and fibroblast growth factor (Fgf) signaling, modulated at least in part by Hip1, controls early lung branching.     

Role of Shh in the development of molecularly characterized tegmental nuclei in mouse rhombomere 1

Hindbrain rhombomeres in general are differentially specified molecularly by unique combinations of Hox genes with other developmental genes. Rhombomere 1 displays special features, including absence of Hox gene expression. It lies within the hindbrain range of the Engrailed genes (En1, En2), controlled by the isthmic organizer via diffusion of FGF8. It is limited rostrally by the isthmus territory, and caudally by rhombomere 2. It is double the normal size of any other rhombomere. Its dorsal part generates the cerebellar hemispheres and its ventral part gives rise to several populations, such as some raphe nuclei, the interpeduncular nucleus, the rhabdoid nucleus, anterior, dorsal, ventral and posterodorsal tegmental nuclei, the cholinergic pedunculopontine and laterodorsal tegmental nuclei, rostral parts of the hindbrain reticular formation, the locus coeruleus, and part of the lateral lemniscal and paralemniscal nuclei, among other formations. Some of these populations migrate tangentially before reaching their final positions. The morphogen Sonic Hedgehog (Shh) is normally released from the local floor plate and underlying notochord. In the present report we explore, first, whether Shh is required in the specification of these r1 populations, and, second, its possible role in the guidance of tangentially migrating neurons that approach the midline. Our results indicate that when Shh function is altered selectively in a conditional mutant mouse strain, most populations normally generated in the medial basal plate of r1 are completely absent. Moreover, the relocation of some neurons that normally originate in the alar plate and migrate tangentially into the medial basal plate is variously altered. In contrast, neurons that migrate radially (or first tangentially and then radially) into the lateral basal plate were not significantly affected.     

Heparan sulfate Ndst1 gene function variably regulates multiple signaling pathways during mouse development

Disruption of heparan sulfate (HS) synthesis in vertebrate development causes malformations that are composites of those caused by mutations of multiple HS binding growth factors and morphogens. We previously reported severe developmental defects of the forebrain and the skull in mutant mice bearing a targeted disruption of the heparan sulfate‐generating enzyme GlcNAc N‐deacetylase/GlcN N‐sulfotransferase 1 (Ndst1). Here, we further characterize the molecular mechanisms leading to frontonasal dysplasia in Ndst1 mutant embryos and describe additional malformations, including impaired spinal and cranial neural tube fusion and skeletal abnormalities. Of the numerous proteins that bind HS, we show that impaired fibroblast growth factor, Hedgehog, and Wnt function may contribute to some of these phenotypes. Our findings, therefore, suggest that defects in HS synthesis may contribute to multifactor types of congenital developmental defects in humans, including neural tube defects. Developmental Dynamics 236:556–563, 2007. © 2006 Wiley‐Liss, Inc.     

Nef-mediated lipid raft exclusion of UbcH7 inhibits Cbl activity in T cells to positively regulate signaling

Lentiviral Nef increases T cell signaling activity, but the molecular nature of the stimulus involved is incompletely described. We explored CD4 T cell lipid raft composition in the presence and absence of Nef. Here, the E2 ubiquitin-conjugating enzyme UbcH7, which acts in conjunction with c-Cbl, is absent from lipid rafts. This Nef-mediated exclusion is associated with failure of ubiquitination of activated Vav. In the presence of Nef, lipid raft Cdc42 is activated and forms a ternary complex between the c-Cbl-interacting protein p85Cool-1/betaPix and c-Cbl, displacing UbcH7 from rafts. Suppression of p85Cool-1/betaPix expression restores UbcH7 raft localization and Vav ubiquitination and diminishes Cdc42 activity. Moreover, p85Cool-1/betaPix knockdown attenuates HIV replication. Thresholds for activation of signaling involve the intricate balance of positive and negative regulators. Here we provide evidence for Nef disruption of a negative regulator of T cell signaling in promoting HIV replication.     

AsialoGM1 and TLR5 cooperate in flagellin-induced nucleotide signaling to activate Erk1/2

Bacterial flagellin can interact with both Toll-like receptor 5 (TLR5) and the cell surface glycolipid, asialoGM1, to activate an innate immune response. The induction of mucin by flagellin in human lung epithelial cells (NCIH292) is dependent on asialoGM1 ligation, ATP receptor signaling, Ca2+ mobilization, and Erk1/2 activation. Conversely, the activation of NF-kappaB by flagellin is dependent on signaling through TLR5. These results prompted us to ask whether the flagellin-induced TLR5 signaling pathway was intersecting with or mutually independent of the nucleotide receptor pathway activated downstream of asialoGM1. Herein, we demonstrate that the release of ATP induced by flagellin is dependent on a Toll signaling cascade. Although Toll was able to activate NF-kappaB in the absence of extracellular ATP, Toll required ATP to activate Erk1/2. These results suggest interdependence between the asialoGM1 and TLR5 pathways and reveal a previously unsuspected role for autocrine extracellular ATP signaling in TLR signaling.     

R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling

In mammals, female development has traditionally been considered a default process in the absence of the testis-determining gene, Sry. Recently, it has been documented that the gene for R-spondin1 (RSPO1), a novel class of soluble activator for Wnt/beta-catenin signaling, is mutated in two Italian families with female-to-male (XX) sex reversal. To elucidate the role of Rspo1 as a candidate female-determining gene in a mouse model, we generated Rspo1-null (Rspo1(-/-)) mice and found that Rspo1(-/-) XX mice displayed masculinized features including pseudohermaphroditism in genital ducts, depletion of fetal oocytes, male-specific coelomic vessel formation and ectopic testosterone production in the ovaries. Thus, although Rspo1 is required to fully suppress the male differentiation program and to maintain germ cell survival during the development of XX gonads, the loss of its activity has proved to be insufficient to cause complete XX sex reversal in mice. Interestingly, these partial sex-reversed phenotypes of Rspo1(-/-) XX mice recapitulated those of previously described Wnt-4(-/-) XX mice. In accordance with this finding, the expression of Wnt-4 and its downstream genes was deregulated in early Rspo1(-/-) XX gonads, suggesting that Rspo1 may participate in suppressing the male pathway in the absence of Sry and maintaining oocyte survival through positively regulating Wnt-4 signaling.     

Sinusoid development and morphogenesis may be stimulated by VEGF‐Flk‐1 signaling during fetal mouse liver development

Early morphogenesis of hepatic sinusoids was histochemically and experimentally analyzed, and the importance of VEGF‐Flk‐1 signaling in the vascular development was examined during murine liver organogenesis. FITC‐gelatin injection experiments into young murine fetuses demonstrated that all primitive sinusoidal structures were confluent with portal and central veins, suggesting that hepatic vessel development may occur via angiogenesis. At 12.5–14.5 days of gestation, VEGF receptors designated Flk‐1, especially their mature form, were highly expressed in endothelial cells of primitive sinusoidal structures and highly phosphorylated on their tyrosine residues. At the same time, VEGF was also detected in hepatoblasts/hepatocytes, hemopoietic cells, and megakaryocytes of the whole liver parenchyma. Furthermore, the addition of VEGF to E12.5 liver cell cultures significantly induced the growth and branching morphogenesis of sinusoidal endothelial cells. Therefore, VEGF‐Flk‐1 signaling may play an important role in the growth and morphogenesis of primitive sinusoids during fetal liver development. Developmental Dynamics 239:386–397, 2010. © 2009 Wiley‐Liss, Inc.     

Fgfr1 regulates development through the combinatorial use of signaling proteins

Fibroblast growth factor (Fgf) signaling governs multiple processes important in development and disease. Many lines of evidence have implicated Erk1/2 signaling induced through Frs2 as the predominant effector pathway downstream from Fgf receptors (Fgfrs), but these receptors can also signal through other mechanisms. To explore the functional significance of the full range of signaling downstream from Fgfrs in mice, we engineered an allelic series of knock-in point mutations designed to disrupt Fgfr1 signaling functions individually and in combination. Analysis of each mutant indicates that Frs2 binding to Fgfr1 has the most pleiotropic functions in development but also that the receptor uses multiple proteins additively in vivo. In addition to Frs2, Crk proteins and Plcγ also contribute to Erk1/2 activation, affecting axis elongation and craniofacial and limb development and providing a biochemical mechanism for additive signaling requirements. Disruption of all known signaling functions diminished Erk1/2 and Plcγ activation but did not recapitulate the peri-implantation Fgfr1-null phenotype. This suggests that Erk1/2-independent signaling pathways are functionally important for Fgf signaling in vivo.     

Shh/Ihh在小鼠胚胎椎间盘形成过程中的表达

文题释义：Shh,Ihh：哺乳动物Hedgehog基因存在3种同源基因,分别为Sonic Hedgehog,India Hedgehog以及Desert Hedgehog,分别编码Shh,Ihh以及Dhh蛋白。它们在胚胎的发育、成体组织和器官干细胞的自我更新与增殖过程中发挥重要的作用。他莫昔芬：是一种选择性雌激素反应调节剂。当向CreER^T2小鼠体内注射他莫昔芬时,由他莫昔芬代谢所产生的雌激素类似物与ERT结合,从而使CreER^T2进入细胞核内发挥Cre重组酶活性。背景：研究表明,Sonic Hedgehog(Shh)在小鼠脊索细胞内有表达,India Hedgehog(Ihh)主要在小鼠胚胎椎体的软骨样细胞以及后期形成的终板中表达。但关于Shh以及Ihh在小鼠胚胎椎间盘形成过程中的表达情况并不清楚。 目的：探究Shh和 Ihh信号在小鼠胚胎椎间盘形成过程中的表达。 方法：将雄性的Shh-CreER^T2; R26-mTmG/+小鼠与雌性R26-mTmG/+小鼠进行交配,获取不同时期(E8.5,E11.5,E12.5,E14.5,E16.5,E18.5)的孕鼠,注射10 g/L的他莫昔芬10 μL/g。当小鼠胚胎处于P0时,取小鼠胚胎椎间盘组织进行基因型鉴定;同时将雄性的C57BL/6小鼠与雌性的C57BL/6小鼠进行交配,获取不同时期(E11.5,E12.5,E14.5,E16.5,E18.5)的孕鼠,取小鼠胚胎椎间盘组织进行分析。通过免疫荧光法检测Shh信号在小鼠胚胎椎间盘形成过程中的表达;免疫组织化学法检测Ihh信号在小鼠胚胎椎间盘形成过程中的表达。所有涉及动物的相关实验均获得苏州大学动物伦理委员会的批准。 结果与结论：①经PCR扩增筛选出基因型为Shh-CreERT2; R26-mTmG/+小鼠胚胎;②免疫荧光染色结果显示,在椎间盘形成过程中,髓核细胞内Shh表达水平逐渐降低;③免疫组织化学染色结果显示,在椎间盘形成过程中,髓核细胞内Ihh表达水平逐渐升高;④结果表明Shh和Ihh在椎间盘形成过程中均发挥调控作用,在一定程度上为研究椎间盘退变的分子机制奠定基础。 ORCID: 0000-0001-9429-2339(巩婷婷) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Expression patterns of ShcD and Shc family adaptor proteins during mouse embryonic development

The Src homology and collagen (Shc) proteins function as molecular adaptors in signaling pathways mediated by a variety of cell surface receptors. Of the four mammalian Shc proteins, ShcD is the least characterized. To this end, ShcD expression was documented and compared to that of other Shc family proteins. In the developing mouse embryo, expression of ShcD overlaps with that of other Shc proteins in the central nervous system, with specific distribution in post‐mitotic neurons. In addition, robust ShcD expression is seen within differentiated epithelial cells of several organs, as well as in skeletal and cardiac muscle, and various tissues of neural crest origin. Interestingly, all Shc family members are expressed in hypertrophic chondrocytes, the first report of Shc protein expression in the developing skeleton. The unique tissue distribution patterns of Shc proteins likely contribute to their complex tissue‐specific signaling functions during embryogenesis. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

CXCR4 and Gab1 cooperate to control the development of migrating muscle progenitor cells

Long-range migrating progenitor cells generate hypaxial muscle, for instance the muscle of the limbs, hypoglossal cord, and diaphragm. We show here that migrating muscle progenitors express the chemokine receptor CXCR4. The corresponding ligand, SDF1, is expressed in limb and branchial arch mesenchyme; i.e., along the routes and at the targets of the migratory cells. Ectopic application of SDF1 in the chick limb attracts muscle progenitor cells. In CXCR4 mutant mice, the number of muscle progenitors that colonize the anlage of the tongue and the dorsal limb was reduced. Changes in the distribution of the muscle progenitor cells were accompanied by increased apoptosis, indicating that CXCR4 signals provide not only attractive cues but also control survival. Gab1 encodes an adaptor protein that transduces signals elicited by tyrosine kinase receptors, for instance the c-Met receptor, and plays a role in the migration of muscle progenitor cells. We found that CXCR4 and Gab1 interact genetically. For instance, muscle progenitors do not reach the anlage of the tongue in CXCR4;Gab1 double mutants; this target is colonized in either of the single mutants. Our analysis reveals a role of SDF1/CXCR4 signaling in the development of migrating muscle progenitors and shows that a threshold number of progenitor cells is required to generate muscle of appropriate size.     

The adapter proteins ADAP and Nck cooperate in T cell adhesion

Nck adapter proteins link receptor and receptor-associated tyrosine kinases with proteins implicated in the regulation of the actin cytoskeleton. Nck is involved in a multitude of receptor-initiated signaling pathways and its physiological role thus covers aspects of tissue development and homeostasis, malignant transformation/invasiveness of tumour cells and also immune cell function. In T cells, changes of cell polarity and morphology associated with cellular activation and effector function crucially rely on the T cell receptor-mediated recruitment and activation of different actin-regulatory proteins to orchestrate and drive cytoskeletal reorganization at the immunological synapse. In a former approach to determine the interactome of Nck in human T cells, we identified the adapter protein ADAP as a Nck-interacting protein. This adhesion and degranulation-promoting adapter protein had already been implicated in the inside-out activation of integrins. Employing co-immunoprecipitations, we demonstrate that both Nck family members Nck1 and Nck2 coprecipitate with ADAP. Specifically, Nck interacts via its Src homology 2 domain with phosphorylated tyrosine Y₅₉₅DDV and Y₆₅₁DDV sites of ADAP. Moreover, we show that endogenous ADAP is phosphorylated in primary human T cell blasts and thus associates with Nck. At the functional level, ADAP and Nck adapter proteins cooperatively facilitate T cell adhesion to the LFA-1 ligand ICAM-1. Our data indicate that the ADAP/Nck complex might provide a means to link integrin activation with the actin cytoskeleton.