Expression of CXCL12 and CXCL14 during eye development in chick and mouse

Vertebrate eye development is a complex multistep process coordinated by signals from the lens, optic cup and periocular mesenchyme. Although chemokines are increasingly being recognized as key players in cell migration, proliferation, and differentiation during embryonic development, their potential role during eye development has not been examined. In this study, we demonstrate by section in situ hybridization that CXCL12 and CXCL14 are expressed during ocular development. CXCL12 is expressed in the periocular mesenchyme, ocular blood vessels, retina, and eyelid mesenchyme, and its expression pattern is conserved between chick and mouse in most tissues. Expression of CXCL14 is localized in the ocular ectoderm, limbal epithelium, scleral papillae, eyelid mesenchyme, corneal keratocytes, hair follicles, and retina, and it was only conserved in the upper eyelid ectoderm of chick and mouse. The unique and non-overlapping patterns of CXCL12 and CXCL14 expression in ocular tissues suggest that these two chemokines may interact and have important functions in cell proliferation, differentiation and migration during eye development.     

MiR-886-3p down regulates CXCL12 (SDF1) expression in human marrow stromal cells

Stromal Derived Factor 1 (SDF1 or CXCL12), is a chemokine known to be critical for the migration of cells in several tissue systems including the homing of the hematopoietic stem cell (HSC) to its niche in the bone marrow. A comparative analysis of miRNA expression profiles of two stromal cell lines, distinguishable by function and by CXCL12 expression (CXCL12+ and CXCL12-), revealed that the CXCL12- cells expressed>40 fold more miR-886-3p than the CXCL12+ cells. Screening studies showed that when miR-886-3p was transfected into the CXCL12+ stromal cells, the expression of CXCL12 was down-regulated by as much as 85% when compared to appropriate controls, and results in the loss of CXCL12-directed chemotaxis. Similar reductions in CXCL12 were obtained with the transfection of miR-886-3p into primary stromal cell cultures. Additional studies showed that miR-886-3p specifically targeted the 3' untranslated region (UTR) of CXCL12 mRNA. These data suggest a role for miRNA in modulating the expression of CXCL12, a gene product with a critical role in hematopoietic regulation.     

Roles for alpha 1 connexin in morphogenesis of chick embryos revealed using a novel antisense approach

Gap junctional communication has been implicated in embryonic development and pattern formation. The gap junction protein, alpha 1 connexin (Cx43) is expressed in dynamic and spatially restricted patterns in the developing chick embryo and its expression correlates with many specific developmental events. High levels of expression are found in regions of budding, which leads to shaping and appears to be a necessary prelude for tissue fusions. In order to investigate the role of alpha 1 connexin in these morphogenetic events, we developed a novel method of applying unmodified antisense deoxyoligonucleotides (ODNs) to chick embryos. The use of pluronic gel to deliver antisense ODNs has allowed us to regulate the expression of alpha 1 connexin protein, both spatially and temporally. This "knockdown" results in some striking developmental defects that mimic some common congenital abnormalities, such as spina bifida, anencephaly, myeloschisis, limb malformation, cleft palate, failure of hematopoiesis, and cardiovascular deformity. The results imply a major role for alpha 1 connexin communication in the integration of signaling required for pattern formation during embryonic development. This novel antisense technique may also be widely applicable.     

Stromal-derived factor-1 (SDF-1) expression during early chick development

Cell migration plays a fundamental role in a wide variety of biological processes including development, tissue repair and disease. These processes depend on directed cell migration along and through cell layers. Chemokines are small secretory proteins that exert their effects by activating a family of G-protein coupled receptors and have been shown to play numerous fundamental roles in the control of physiological and pathological processes during development and in adult tissues, respectively. Stromal-derived factor-1 (SDF-1/CXCL12), a ligand of the chemokine receptor, CXCR4, is involved in providing cells with directional cues as well as in controlling their proliferation and differentiation. Here we studied the expression pattern of SDF-1 in the developing chick embryo. We could detect a specific expression of SDF-1 in the ectoderm, the sclerotome, the intersomitic spaces and the developing limbs. The expression domains of SDF-1 reflect its role in somitic precursor migration and vessel formation in the limbs.     

Role of FK506-binding protein 12 in development of the chick embryonic heart

A cDNA encoding chicken FK506-binding protein 12 (FKBP12) was isolated and sequenced. The predicted amino acid sequence of the chicken protein shows high homology to those of FKBP12 proteins of other species ranging from human to frog. The possible role of FKBP12 in chick embryonic cardiac development was examined. Northern blot analysis revealed that FKBP12 mRNA is distributed widely in chick embryos, being especially abundant in the heart; the amount of FKBP12 mRNA in the embryonic heart decreased with time. Administration of FK506 to chick embryos at 7 to 9 days resulted in marked cardiac enlargement. FK506 also reduced the expression of myosin, induced a more elongated cell morphology, and impaired network formation in cultured chick embryonic cardiomyocytes. These results suggest that FKBP12 is important in the regulation of contractile function and phenotypic expression in chick cardiomyocytes during embryonic development.     

CXCR4 regulates migration of lung alveolar epithelial cells through activation of Rac1 and matrix metalloproteinase-2

Restoration of the epithelial barrier following acute lung injury is critical for recovery of lung homeostasis. After injury, alveolar type II epithelial (ATII) cells spread and migrate to cover the denuded surface and, eventually, proliferate and differentiate into type I cells. The chemokine CXCL12, also known as stromal cell-derived factor 1α, has well-recognized roles in organogenesis, hematopoiesis, and immune responses through its binding to the chemokine receptor CXCR4. While CXCL12/CXCR4 signaling is known to be important in immune cell migration, the role of this chemokine-receptor interaction has not been studied in alveolar epithelial repair mechanisms. In this study, we demonstrated that secretion of CXCL12 was increased in the bronchoalveolar lavage of rats ventilated with an injurious tidal volume (25 ml/kg). We also found that CXCL12 secretion was increased by primary rat ATII cells and a mouse alveolar epithelial (MLE12) cell line following scratch wounding and that both types of cells express CXCR4. CXCL12 significantly increased ATII cell migration in a scratch-wound assay. When we treated cells with a specific antagonist for CXCR4, AMD-3100, cell migration was significantly inhibited. Knockdown of CXCR4 by short hairpin RNA (shRNA) caused decreased cell migration compared with cells expressing a nonspecific shRNA. Treatment with AMD-3100 decreased matrix metalloproteinase-14 expression, increased tissue inhibitor of metalloproteinase-3 expression, decreased matrix metalloproteinase-2 activity, and prevented CXCL12-induced Rac1 activation. Similar results were obtained with shRNA knockdown of CXCR4. These findings may help identify a therapeutic target for augmenting epithelial repair following acute lung injury.     

Cloning and expression of the Wnt antagonists Sfrp-2 and Frzb during chick development

The Wnt genes are known to play fundamental roles during patterning and development of a number of embryonic structures. Receptors for Wnts are members of the Frizzled family of proteins containing a cysteine-rich domain (CRD) that binds the Wnt protein. Recently several secreted frizzled-related proteins (Sfrps) that also contain a CRD have been identified and some of these can both bind and antagonise Wnt proteins. In this paper we report the expression patterns of the chick homologues of Frzb, a known Wnt antagonist, and Sfrp-2. Both genes are expressed in areas where Wnts are known to play a role in development, including the neural tube, myotome, cartilage, and sites of epithelial-mesenchymal interactions. Initially, Sfrp-2 and Frzb are expressed in overlapping areas in the neural plate and neural tube, whereas later, they have distinct patterns. In particular Sfrp-2 is associated with myogenesis while Frzb is associated with chondrogenesis, suggesting that they play different roles during development. Finally, we have used the early Xenopus embryo as an in vivo assay to show that Sfrp-2, like Frzb, is a Wnt antagonist. These results suggest that Sfrp-2 and Frzb may function in the developing embryo by modulating Wnt signalling.     

Analysis of <Emphasis Type="Italic">EphA4</Emphasis> in the lesser spotted catshark identifies a primitive gnathostome expression pattern and reveals co-option during evolution of shark-specific morphology

The Eph family is the largest known group of structurally related receptor tyrosine kinases (RTKs). Each Eph receptor has a specific Ephrin ligand, and these function to define spatial boundaries during development. Analyses of EphA4 in mouse, chick, frog and zebrafish embryos have implicated this gene in a number of developmental processes, including maintenance of segmental boundaries, axon guidance, limb development, neural crest migration and patterning of the ear. In order to determine which components of EphA4 function may be primitive for gnathostomes, we cloned EphA4 from the lesser spotted catshark (Scyliorhinus canicula) and examined its expression pattern during shark embryonic development. Consistent with the patterns reported for bony fish and tetrapods, we observed segmental expression of EphA4 in the developing hindbrain and later in the pharyngeal arches of shark embryos. EphA4 was also detected during sensory organogenesis, in the developing ear, eye, nasal pits and lateral line. A dynamic pattern of EphA4 expression occurs during shark fin development, suggesting an early role in outgrowth and patterning of the fin buds and a later role in tissue differentiation. We also observed several novel domains of EphA4 expression that have not been reported in other vertebrates, including external gill buds, dermal denticles, median fins and claspers. While some of these domains may reflect co-option of EphA4 expression to novel sites for development of shark-specific characters, others are more likely to be ancestral patterns of expression that were lost in other vertebrate lineages.Edited by R. P. Elinson     

CXCL14 inhibits trophoblast outgrowth via a paracrine/autocrine manner during early pregnancy in mice

CXCL14, a member of chemokine family, was previously known to participate in many pathophysiological events, such as leukocytes recruitment and tumor suppression. However, it remained largely unknown whether CXCL14 is a physiological player during early pregnancy. In this regard, our recent global gene microarray analysis has observed an implantation-specific expression profile of CXCL14 mRNA during early pregnancy in mice, showing its higher levels at implantation sites compared to inter-implantation sites, implicating a potential role of CXCL14 in the periimplantation events. In the present investigation, using Northern blot, in situ hybridization and immunostaining, we further demonstrated that uterine CXCL14 expression was specifically induced at embryo implantation site and expanded with subsequent decidualization process in a spatiotemporal manner. The implanting embryo also showed a highlighted expression of CXCL14 in the blastocyst trophectoderm and its derived ectoplacental cones (EPCs) during postimplantation development. In vitro functional study revealed that CXCL14 could significantly inhibit both primary and secondary trophoblast attachment and outgrowth, correlated with a stage-dependant downregulation of MMP-2 and/or MMP-9 activity. Moreover, it was found that biotinylated CXCL14 could specifically bind to trophoblast cells in vitro and in vivo, suggesting trophoblast cell, perhaps expressing the unidentified CXCL14 receptor, is a bioactive target of CXCL14. Collectively, our findings provide evidences supporting the contention that CXCL14 is an important paracrine/autocrine modulator regulating trophoblast outgrowth at the maternal–fetal interface during the process of pregnancy establishment. This study is clinically related since CXCL14 is also highly expressed in human receptive endometrium and trophoblasts. J. Cell. Physiol. 221: 448–457, 2009. © 2009 Wiley-Liss, Inc.     

Hemodynamic-dependent patterning of endothelin converting enzyme 1 expression and differentiation of impulse-conducting Purkinje fibers in the embryonic heart

Impulse-conducting Purkinje fibers differentiate from myocytes during embryogenesis. The conversion of contractile myocytes into conduction cells is induced by the stretch/pressure-induced factor, endothelin (ET). Active ET is produced via proteolytic processing from its precursor by ET-converting enzyme 1 (ECE1) and triggers signaling by binding to its receptors. In the embryonic chick heart, ET receptors are expressed by all myocytes, but ECE1 is predominantly expressed in endothelial cells of coronary arteries and endocardium along which Purkinje fiber recruitment from myocytes takes place. Furthermore, co-expression of exogenous ECE1 and ET-precursor in the embryonic heart is sufficient to ectopically convert cardiomyocytes into Purkinje fibers. Thus, localized expression of ECE1 defines the site of Purkinje fiber recruitment in embryonic myocardium. However, it is not known how ECE1 expression is regulated in the embryonic heart. The unique expression pattern of ECE1 in the embryonic heart suggests that blood flow-induced stress/stretch may play a role in patterning ECE1 expression and subsequent induction of Purkinje fiber differentiation. We show that gadolinium, an antagonist for stretch-activated cation channels, downregulates the expression of ECE1 and a conduction cell marker, Cx40, in ventricular chambers, concurrently with delayed maturation of a ventricular conduction pathway. Conversely, pressure-overload in the ventricle by conotruncal banding results in a significant expansion of endocardial ECE1 expression and Cx40-positive putative Purkinje fibers. Coincident with this, an excitation pattern typical of the mature heart is precociously established. These in vivo data suggest that biomechanical forces acting on, and created by, the cardiovascular system during embryogenesis play a crucial role in Purkinje fiber induction and patterning.     

CXCL14 is no direct modulator of CXCR4

C-X-C motif chemokine 12/C-X-C chemokine receptor type 4 (CXCL12/CXCR4) signaling is involved in ontogenesis, hematopoiesis, immune function and cancer. Recently, the orphan chemokine CXCL14 was reported to inhibit CXCL12-induced chemotaxis – probably by allosteric modulation of CXCR4. We thus examined the effects of CXCL14 on CXCR4 regulation and function using CXCR4-transfected human embryonic kidney (HEK293) cells and Jurkat T cells. CXCL14 did not affect dose–response profiles of CXCL12-induced CXCR4 phosphorylation, G protein-mediated calcium mobilization, dynamic mass redistribution, kinetics of extracellular signal-regulated kinase 1 (ERK1) and ERK2 phosphorylation or CXCR4 internalization. Hence, essential CXCL12-operated functions of CXCR4 are insensitive to CXCL14, suggesting that interactions of CXCL12 and CXCL14 pathways depend on a yet to be identified CXCL14 receptor.     

Pleiotropic functions of the CXC-type chemokine CXCL14 in mammals

CXCL14 is a member of the CXC chemokine family. CXCL14 possesses chemoattractive activity for activated macrophages, immature dendritic cells and natural killer cells. CXCL14-deficient mice do not exhibit clear immune system abnormalities, suggesting that the function of CXCL14 can be compensated for by other chemokines. However, CXCL14 does appear to have unique biological roles. It suppresses the in vivo growth of lung and head-and-neck carcinoma cells, whereas the invasiveness of breast and prostate cancer cells appears to be promoted by CXCL14. Moreover, recent evidence revealed that CXCL14 participates in glucose metabolism, feeding behaviour-associated neuronal circuits, and anti-microbial defense. Based on the expression patterns of CXCL14 and CXCL12 during embryonic development and in the perinatal brain in mice, the functions of these two chemokines may be opposite or interactive. Although CXCL14 receptors have not yet been identified, the intracellular activity of CXCL14 in breast cancer cells suggests that the CXCL14 receptor(s) and signal transduction pathway(s) may be different from those of conventional CXC-type chemokines.     

Expression of ASK1 during chick and early mouse development

Apoptosis signal-regulating kinase 1 (ASK1) is an important regulator of stress-induced cell death. ASK1 is activated by oxidative stress, TNF and endoplasmatic reticulum stress and activates the JNK- and p38-dependent intracellular death pathways. A number of studies have suggested that ASK1 may also have other roles in addition to its pro-apoptotic activity. Expression of ASK1 during early embryonic development has so far not been analyzed. We have identified and cloned chick ASK1 in a screen for FGF8 inducible genes in chick facial mesenchyme. Here we report the expression of chick ASK1 from the gastrulation stage (HH4) to day 4 of development, its expression in the developing inner organs and limbs, and we compare its expression to the expression of Ask1 during mouse development. Furthermore, we provide evidence that FGF signaling is required for ASK1 expression in chick nasal mesenchyme. In contrast, expression in the mouse nasal region was restricted to the epithelium and was independent of FGF signaling. Our analysis demonstrates that ASK1 has a spatially restricted and temporally dynamic expression pattern in both chick and mouse embryos, which includes conserved as well as species-specific expression domains.