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Keywords:

  • SOCS3;
  • cytokine signaling;
  • wounding;
  • MAP kinase

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The suppressor of cytokine signaling (SOCS) family of proteins are intracellular mediators of cytokine signaling. These proteins are induced rapidly by cytokine stimulation and act in a classic negative-feedback loop to attenuate the cellular response to the cytokine signal. In this study, we present the cloning and initial characterization of the Xenopus SOCS3 gene. We show that xSOCS3 is rapidly induced in response to epithelial wounding in the tadpole. The induction of xSOCS3 in response to trauma is transient with maximal expression being reached 1 hr after the injury and diminishing after that. Unlike other genes known to be responsive to wound-induced activation of the mitogen-activated protein (MAP) kinase pathway, such as Egr1, SOCS3 expression in response to trauma is unaffected by blockade of the MAP kinase pathway by chemical inhibitors. Developmental Dynamics 233:1123–1130, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Cytokines play an important role in vertebrate homeostasis by controlling the growth and differentiation of target cells. Binding of these extracellular ligands to transmembrane receptors at the surface of cells initiates signal transduction pathways that influence several cellular processes. The components of the downstream signal pathways that transduce these signals have been the focus of intense study in recent years. Interaction of a cytokine with its receptor results in receptor aggregation and activation of accessory protein kinase proteins called Janus kinases (JAKs). Activation of JAK tyrosine kinases results in the phosphorylation of specific residues in the intracellular domain of the cytokine receptor that, in turn, act as docking sites for proteins that initiate multiple signal transduction cascades, including phosphatidylinositol 3-kinase (PI3K), ras, phospholipase C-γ, and the signal transducers and activators of transcription (STATs; O'Shea et al., 2002). An important component of the intracellular effector cascades are the negative regulatory molecules that serve to limit the response of the cell to the cytokine signal (Krebs and Hilton, 2001). Three classes of signal modulators that inhibit the cytokine-mediated signaling pathways have been identified: the protein tyrosine phosphatases, the protein inhibitors of activated STATs (PIAS) and the suppressor of cytokine signaling (SOCS) proteins.

There are eight known members of the SOCS family (SOCS1–7 and CIS; Krebs and Hilton, 2001). The SOCS family is defined by the presence of a Src-homology 2 domain (binds proteins at the sites of phosphorylated tyrosine residues) and a SOCS box (a 40 amino acid domain in the carboxyl terminal domain). The SOCS proteins act in a negative-feedback loop to suppress signal transduction from cytokine receptors by at least two distinct mechanisms. SOCS1, for example, binds to JAK proteins and inhibits their kinase activity. CIS uses a different mechanism to inhibit cytokine signaling, it directly associates with the cytokine receptor and competes with STAT proteins for activated binding sites. SOCS3 also binds to the cytokine receptor but blocks Janus kinase activity by binding JAK-proximal sites on the activated receptor. The SOCS proteins are induced by cytokine receptor signaling pathways. For example, SOCS3, the focus of this study, is induced by a variety of cytokine signals, including interleukin (IL) -2, IL-3, IL-4, IL-6, IL-10, interferon-γ (IFN-γ), erythropoietin (EPO), prolactin, granulocyte/macrophage colony stimulating factor (GM-CSF), G-CSF, leukemia inhibitory factor (LIF), growth hormone (GH), and lipopolysaccharide. The induction of SOCS expression by cytokine signaling is thought to be mediated, in part, by the binding of STAT proteins to the proximal promoter regions of the SOCS genes. SOCS proteins can bind and inhibit multiple cytokine receptors and their associated molecules, thus providing potential crosstalk for multiple cytokine signaling pathways. For example, SOCS3 binds and inhibits signaling from Lck, gp130, GH receptor, EPO receptor, leptin receptor, insulin-like growth factor-1 receptor, PYK2, and the fibroblast growth factor receptor. Targeted gene disruption experiments in mice have shown that SOCS3 is essential for embryonic survival (Marine et al., 1999; Roberts et al., 2001).

The isolation of Xenopus cytokine receptors has been hampered by the low degree of sequence homology with mammalian counterparts. We sought to identify intracellular components of the cytokine signaling pathways. In this study, we present the cloning and initial characterization of the Xenopus SOCS3 (xSOCS3) protein. To our surprise, in situ hybridization studies revealed that xSOCS3 is rapidly induced at the site of epithelial wounds. The induction of SOCS3 by epithelial wounding is transient, with maximal expression being reached 1 hr after trauma and diminishing thereafter.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Isolation and Characterization of Xenopus SOCS3 Sequences

A full-length Xenopus laevis xSOCS3 cDNA clone was identified by an in silico screen of the expressed sequence tag (EST) databases using the murine SOCS3 sequence as a probe. Sequence alignments showed that the Xenopus laevis and Xenopus tropicalis SOCS3 proteins share extensive sequence similarity with avian and mammalian SOCS3 proteins (Fig. 1A,B). The highest levels of sequence conservation are seen in the src-homology-2 (SH2) domain and in the SOCS box. The Xenopus tropicalis sequence was predicted from a genomic sequencing scaffold that contains the SOCS3 gene (Fig. 1C). Like other vertebrate SOCS3 genes, the X. tropicalis consists of two exons and a single intron that lies in the 5′ untranslated region of the transcript. Transcription factor binding site prediction software (TESS) indicated that three STAT-binding sites are located in the proximal promoter of the X. tropicalis SOCS3 gene. STAT bindings sites have been identified in the promoter regions of mammalian CIS, SOCS1, and SOCS3 genes.

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Figure 1. A: Sequence alignment of vertebrate suppressor of cytokine signaling-3 (SOCS3) proteins. (Accession numbers for sequences used to generate this alignment are Human O14543, Cow C9BEG9, Chicken C90X67, Mouse NP_031733, Rat NP_446017.) B: Phylogenetic tree of SOCS3 protein sequences indicates the close evolutionary relationship between the vertebrate orthologs. C: Genomic sequence surrounding the Xenopus tropicalis SOCS3 gene. The sequence shown represents nucleotides 270,081 to 272,720 of scaffold 14 (2,497,805 bp) of the Joint Genome Institute (JGI) version 2 release of the Xenopus tropicalis genomic sequencing project. The entire open reading frame is located on exon 2 and is depicted in single letter code beneath the corresponding nucleotide sequence. The single intron (lower case type) is located in the 5′ untranslated region (UTR) of the predicted mRNA. Alignment of the genomic sequence and that of a X. tropicalis SOCS3 EST clone (IMAGE 6998184, 5′ UTR sequence is underlined) confirmed the positions of the exon/intron boundaries. Analysis of this region using a transcription factor binding site algorithm (TESS; www.cbil.upenn.edu) predicts three STAT binding sites in the proximal promoter region of the X. tropicalis SOCS3 gene (boldface and underlined).

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Expression of SOCS3 During Embryonic Development

The developmental expression of SOCS3 was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. PCR amplification of SOCS3 sequences from cDNA prepared from total cellular RNA from staged embryos indicates that SOCS3 is not present as a maternal transcript. Zygotic expression of SOCS3 begins at late gastrula (stage 12) and remains at a relatively constant level throughout early development (stages 12–45; Fig. 2).

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Figure 2. Developmental expression of suppressor of cytokine signaling-3 (SOCS3). Total cellular RNA from staged embryos was used as to produce first-strand cDNA template for the amplification reaction. Ornithine decarboxylase (ODC) was used as control for RNA recovery. Numbers represent the Nieuwkoop and Faber stage of development. “no RNA” is a negative control that lacked template.

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In situ hybridization studies using antisense riboprobes indicated very low (barely detectable) levels of xSOCS3 expression in the ventral region of tail bud embryos (data not shown). In late neurulae and tail bud stages (stage 18 to 24), intense random spots of SOCS3 expression were detected in approximately 10% of the embryos. At this stage in development, the embryos are removed from the vitelline membrane and allowed to uncurl before fixing for in situ hybridization. We hypothesized that the random spots on these embryos could be due to small wounds inflicted during the manual removal of the membranes. To test this hypothesis, groups of late neurula embryos, while still inside the vitelline membrane, were pricked with a needle on the left flank and allowed to heal for 10 and 30 min before fixing for in situ analysis. Intense SOCS3 expression was detected at the site of wounding in 100% of the embryos (n = 61) that were allowed to heal for 30 min before fixing (data not shown and Fig. 3A). Embryos that healed for 10 min before fixing showed very faint staining at the site of injury. To ensure that the staining was not due to nonspecific trapping of the probe at the site of injury, embryos were fixed first and then subjected to a similar postmortem injury. None of the prefixed then wounded embryos showed staining at the site of the injury (data not shown). Likewise, injuries followed by healing times of less than 2 min did not show staining with the SOCS3 antisense riboprobe (data not shown). To examine the time course of SOCS3 expression after injury, we allowed embryos to heal for 10, 30, 60, 120 (2 hr), 240 (4 hr), and 480 (8 hr) min after injury. As shown in Figure 3A, the expression of SOCS3 is maximal after healing for 60 min after a superficial epithelial wound. Two hours after the injury, the levels of SOCS3 staining had diminished; by 4 hr the staining was barely detectable and was not present after 8 hr (Fig. 3A). To determine whether this phenomenon was also present at other stages of development, we injured embryos at different stages, starting at gastrulation through swimming tadpole stages, allowed them to heal for 1 hr, and then fixed the samples for in situ analysis. As shown in Figure 3B, injury at the early gastrula stage results in robust expression of SOCS3 1 hr after the trauma (Fig. 3B and data not shown). These data show that SOCS3 expression is induced rapidly, but transiently, after epithelial trauma in the developing Xenopus embryo.

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Figure 3. Suppresor of cytokine signaling-3 (SOCS3) expression is rapidly induced after epithelial wounding. A: Embryos were subjected to superficial epithelial wounds, allowed to heal for the stated times (0–8 hr) and then subjected to in situ hybridization using a SOCS3 antisense riboprobe. SOCS3 is first evident at 30 min after trauma and is maximal at 1 hr. The wound-induced expression of SOCS3 is transient and dramatically down-regulated 2 hr after trauma and is undetectable by 8 hr after trauma. B: SOCS3 is rapidly induced after trauma at early stages of embryonic development. Embryos at the gastrula stage (stage 11) were wounded and allowed to heal for 1 hr before fixation and in situ hybridization for SOCS3 expression. C: SOCS3 expression is induced in the epidermis and underlying tissues after trauma. Cross-section (8 μm) of a stage 28 embryo after wounding, healing for 1 hr, and then analyzed for SOCS3 expression by in situ hybridization. SOCS3 expression (blue staining) in evident in the epidermis and the underlying tissues. Arrow indicates the site of wounding, and the parallel dotted lines indicate the depth of the epidermal layer. Original magnification, ×200. N, notochord; S, somite; fg, foregut; e, epidermis.

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To determine whether the expression of SOCS3 after trauma extends below the epidermis, we performed histological analysis of embryos after in situ hybridization. Figure 3C clearly indicates that the expression of SOCS3 is not limited to the epidermis but is expressed in the underlying tissues as well.

Blocking ERK Kinase Activation Does Not Inhibit Induction of SOCS3 Expression After Epithelial Wounding

The SOCS proteins act as negative-feedback inhibitors of intracellular signaling cascades. As their name suggests, the SOCS proteins are involved in suppression of cytokine signaling cascades by directly binding the upstream components of the cytokine signal transduction pathways. In an effort to determine the signaling pathway that up-regulates the expression of SOCS3 after injury, we used chemical inhibitors of known components of the wounding response. Activation of the mitogen-activated protein (MAP) kinase pathway has been well described in Xenopus epithelial wounding models (LaBonne and Whitman, 1997; Christen and Slack, 1999). Immunohistochemistry for activated extracellular signal-regulated kinase (ERKPP, bis-phosphorylated ERK) demonstrates rapid induction of the MAP kinase pathway after epithelial wounding in Xenopus (Fig. 4A). Within minutes after an epithelial trauma, there is robust staining for activated ERK at the site of the injury. The activation of the ERK protein extends further from the site of injury than does the expression of SOCS3, suggesting that the SOCS3-expressing cells around the injury site are a subset of the ERK-responding cells (compare Fig. 4A and C). The activation of ERK can be blocked by a pretreatment of the embryos with a MEK inhibitor (PD98059; 50 μM final concentration). Inhibition of the MAP kinase pathway at this point blocks the phosphorylation (and activation) of the downstream substrate ERK. Pretreatment of the embryos with either a p38 inhibitor (SB202190; 50 μM final concentration) or a JAK2 inhibitor (AG490; 50 μM final concentration) did not lead to suppression of ERK activation after injury (Fig. 4B). To determine whether suppression of the MAP kinase pathway perturbed the induction of SOCS3 at the site of epithelial trauma, we pretreated embryos with the same inhibitors and measured SOCS3 expression by in situ hybridization 1 hr after the trauma. The blockade of the MAP kinase pathway by the MEK inhibitor PD98059 had no effect on the induction of SOCS3 expression at the site of injury. This finding indicates that the induction of SOCS3 after epithelial trauma is independent of a functioning MAP kinase pathway. Similarly, pretreatment with the JAK2 inhibitor or the p38 inhibitor had no effect on the induction of SOCS3 expression after trauma. Combinations of the MEK inhibitor (PD98059) with either the JAK2 inhibitor (AG490) or the p38 inhibitor (SB202190) had no effect on the induction of SOCS3 expression after trauma (data not shown). This finding is in contrast to other genes known to be up-regulated during the early stages of wound healing. For example, early growth response (Egr)-1 (also known as Krox-24, NGF1-A, zif268, and tis8) is up-regulated rapidly but transiently at the site of wounding (Dieckgraefe and Weems, 1999; Grose et al., 2002). In an in vitro model system, wounding of tissue culture epithelial monolayers resulted in significant induction of activated ERK and expression of Egr-1. ERK activation and Egr-1 expression were both inhibited in a dose-dependent manner by the MEK inhibitor PD98059. Induction of c-fos expression was also partially, but not completely, blocked by the application of the MEK inhibitor (Dieckgraefe and Weems, 1999). Taken together, this evidence suggests that the induction of SOCS3 after epithelial trauma is independent of the pathways that result in Egr-1 expression.

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Figure 4. A: Extracellular signal-regulated kinase (ERK) is activated rapidly after epithelial trauma. Embryos were wounded and allowed to heal for 2 or 10 min before fixation and immunohistochemistry for activated (bis-phosphorylated) ERK. ERK activation is evident within 2 min after trauma and is extensive after 10 min. B: Trauma-induced ERK activation is blocked by the mitogen-activated protein/ERK kinase (MEK1) inhibitor PD98059. Embryos were pretreated for 2 hr in 0.1× MMR containing either PD98059 or AG490 or SB202190 (each at 50 μM final concentration; see Table 1) before trauma. After 10 min, the embryos were fixed and immunostained for activated ERK. Inhibition of MEK1 by PD98059 resulted in a block in ERK activation after injury. Inhibition of JAK2 (AG490) or p38 (SB202190) did not affect the activation of ERK after trauma. C: Wound-induced SOCS3 expression is not inhibited by blocking MEK1 activity. Embryos were pretreated for 2 hr in 0.1× MMR containing either PD98059 or AG490 or SB202190 (each at 50 μM final concentration; see Table 1) before trauma. After 1 hr, the embryos were fixed and SOCS3 expression was assayed by in situ hybridization. Blocking MEK1 activity had no affect on the induction of SOCS3 expression at the site of epithelial wounding.

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Table 1. Chemical Inhibitors Used to Inhibit Wound-Induced Activation of SOCS3 Expression
InhibitorFunctionFinal concentration usedaPretreatment time (min)
  • a

    Final concentrations used were as recommended by the supplier (Calbiochem, CA).

  • b

    Longer pretreatment times resulted in death of the embryos before the end of the recovery period (1 hour after trauma).

  • c

    Recommended dilution of the cocktail of phosphatase inhibitors.

  • SOCS 3, suppressor of cytokine signaling-3; MEK, MAP/ERK kinase; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; DMBI, {(Z)-3-[4-(Dimethylamino)benzylidenyl]indolin-2-one}; MAP, mitogen-activated protein; PDGF, platelet-derived growth factor; EGFR, epidermal growth factor receptor; EGTA, ethyleneglycoltetraacetic acid; PI, phosphatase inhibitor.

PD98059MEK1 inhibitor50 μM120
AGE490EGF receptor kinase, JAK2/3 inhibitor50 μM120
SB202190p38 inhibitor, no activity on ERK50 μM120
AutocamtideCell-permeable calmodulin-dependent protein kinase II (CamKII) inhibitor5 μM120
DMBIPDGFβ receptor kinase inhibitor, does not inhibit EGFR or c-src tyrosine kinases50 μM120
IonomycinMobile carrier of calcium ions5 nM or 50 nM120
GenisteinProtein tyrosine kinase inhibitor50 μM120
GenistinInactive analog of Genistein50 μM120
EGTAChelates calciums ions0.5 mM and 2 mM10b
BpV (biyp)Phosphotyrosine phosphatase inhibitor100 nM120
AG1296PDGF receptor kinase inhibitor1 μM120
PP2Src family tyrosine kinase inhibitor10 nm120
Y276632Rho-associated protein kinases and ROCK-II inhibitor140 nM120
SU6656Src family kinases, fyn, lyn, and yes300 nM120
Rafl kinase inhibitorc-Rafl kinase inhibitor10 nM120
PI cocktail set IIInhibits dephosphorylation reactions1:100 dilutionc120

We tested whether ectopic expression of SOCS3 mRNA could attenuate the activation of the MAP kinase pathway in whole embryos. Embryos were injected at the two-cell stage in both blastomeres with SOCS3 mRNA (1 ng), allowed to develop to gastrula stage (stage 11), and wounded with a sharp needle. Immunohistochemistry for activated ERK was performed 10 min after wounding. The ectopic expression of SOCS3 mRNA had no effect on the induction of ERK activation after trauma (data not shown).

To test other signaling pathways that may regulate the expression of SOCS3 after trauma, we tested other chemical inhibitors in our SOCS3 in situ hybridization assay. A list of the inhibitors that were tried and the molecules that they target is presented in Table 1. None of the chemical inhibitors used had any affect on the induction of SOCS3 after epithelial trauma (data not shown). An important caveat to note is that, with the exception of the MEK inhibitor PD98059, we do not have internal controls to test that the inhibitors are functioning as predicted in the whole embryo model.

Other investigators have implicated SOCS3 as an injury and stress response gene. In mice subjected to transverse aortic constriction (TAC) leading to biomechanical stress of pressure overload, SOCS3 levels in cardiomyocytes increased dramatically in a biphasic manner. The first peak of SOCS3 expression occurred during the acute response phase (1 to 3 hr after the onset of TAC), and the second peak occurred during the hypertrophic response phase at 2 days after TAC. Ectopic expression of SOCS3 in cardiomyocytes was shown to attenuate multiple signaling pathways downstream of the gp130 receptor, including phosphorylation of STAT-3 and activation of AKT, MEK-1, and ERK1/2 (Yasukawa et al., 2001). Using a cDNA microarray approach, SOCS3 was also found to be up-regulated during the healing phase (18–22 hr after trauma) in mouse corneas after laser ablation (Cao et al., 2002). In a thermal injury model, SOCS3 expression increases in the liver of rats that receive a third-degree skin burn (Ogle et al., 2000; Kong et al., 2002).

Our results show that SOCS3 is a component of the wounding response in embryonic vertebrate epithelia. SOCS3 now joins a growing list of genes that are induced during the acute phase of the wounding response, including Egr-1, xwig, and fos (Dieckgraefe and Weems, 1999; Klingbeil et al., 2001). It is likely that the expression of SOCS3 at the site of wounding serves to attenuate the signaling pathways that are initiated by trauma and, thus, protect the responding cells from hyperactivation of these pathways.

Future studies will address the role of SOCS3 in the healing process. We will determine the signaling pathway(s) that lead to SOCS3 expression after trauma and investigate the function of SOCS3 in the wounding response.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Identification of Xenopus SOCS3 Sequences

Three full-length Xenopus laevis SOCS3 cDNA clones were identified by virtual screening of the EST databases using the mouse SOCS3 sequence (GenBank accession nos. BG017633, BC068752, and BG234045). The clones were obtained from the RZPD and fully sequenced. These clones shared 100% and 95% identity with two recently annotated Xenopus laevis clones MGC:81248 and SOCS-prov, respectively (accession nos. AAH68752 and AAH54214, respectively; Klein et al., 2002). The Xenopus laevis sequence was used to identify Xenopus tropicalis genomic sequences in the Joint Genome Institute (JGI) database. A contig of overlapping sequences revealed that the entire open reading frame of Xenopus tropicalis SOCS3 is encoded on a single exon. The X. tropicalis SOCS3 gene is encoded on scaffold_14 (2,497,805 bp) of the JGI X. tropicalis assembly version 2. A single Xenopus tropicalis SOCS3 EST was identified (IMAGE 6998184, accession no. CF346281).

RT-PCR and In Situ Hybridization

Total RNA was harvested from staged embryos using Qiaquik RNA extraction kits (Qiagen, CA) according to manufacturers instructions. RT-PCR was performed as described (Kelley et al., 1994). Radiolabeled products were resolved on polyacrylamide gels (Criterion precast gels, Bio-Rad) and subjected to autoradiography. Xenopus SOCS3 primers used for RT-PCR were XSOCS3U, 5′ CCG GTG CCT AGG AAG AAG A; XSOCS3L, 5′ CCT TAA AGC AGC CGC CAT C.

Ornithine decarboxylase (ODC) was used to control for RNA recovery. ODC primers are described on the Xenbase Web site. In situ hybridization was performed according to the method described by Harland (1991).

Embryo Manipulations

Embryos were fertilized in vitro, the jelly coat removed by soaking in cysteine (3% w/v in 0.1× MMR for 3 min), and maintained in 0.1× MMR at 18–20°C until the desired stage was reached (Sive et al., 1998). Small epithelial wounds were induced in embryos using either a tungsten needle or a single point of a pair of biological grade forceps (Dupont #55). Inhibitors and controls were added to the incubation medium (0.1× MMR) 2 hr before injury to allow for absorption by the embryo. The embryos remained in the medium containing the inhibitors during the recovery period (1 hr). Embryos were staged according to the normal tables of Nieuwkoop and Faber (1994).

Synthetic xSOCS3 mRNA was produced using mMessage mMachine RNA synthesis kits (Ambion) according to the manufacturer's instructions. RNA concentration was measured by spectrophotometric analysis, and the integrity of the sample was determined by electrophoresis.

Histology

After in situ hybridization, embryos were dehydrated in ethanol and solvent exchanged into xylene and then embedded in paraffin overnight at 56°C. Cross-sections were taken at 8 μm, dewaxed with xylene, and cover-slipped with no counterstaining.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

We thank members of the MeadLab for helpful discussions and comments on the manuscript. We thank Dr. Clair Kelley and Evan Parganas for critical reading of the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  • Cao Z, Wu HK, Bruce A, Wollenberg K, Panjwani N. 2002. Detection of differentially expressed genes in healing mouse corneas, using cDNA microarrays. Invest Ophthalmol Vis Sci 43: 28972904.
  • Christen B, Slack JM. 1999. Spatial response to fibroblast growth factor signalling in Xenopus embryos. Development 126: 119125.
  • Dieckgraefe BK, Weems DM. 1999. Epithelial injury induces egr-1 and fos expression by a pathway involving protein kinase C and ERK. Am J Physiol 276: G322G330.
  • Grose R, Harris BS, Cooper L, Topilko P, Martin P. 2002. Immediate early genes krox-24 and krox-20 are rapidly up-regulated after wounding in the embryonic and adult mouse. Dev Dyn 223: 371378.
  • Harland RM. 1991. In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol 36: 685695.
  • Kelley C, Yee K, Harland R, Zon LI. 1994. Ventral expression of GATA-1 and GATA-2 in the Xenopus embryo defines induction of hematopoietic mesoderm. Dev Biol 165: 193205.
  • Klein SL, Strausberg RL, Wagner L, Pontius J, Clifton SW, Richardson P. 2002. Genetic and genomic tools for Xenopus research: the NIH Xenopus initiative. Dev Dyn 225: 384391.
  • Klingbeil P, Frazzetto G, Bouwmeester T. 2001. Xwig1, a novel putative endoplasmic reticulum protein expressed during epithelial morphogenesis and in response to embryonic wounding. Int J Dev Biol 45: 379385.
  • Kong F, Guo X, Noel JG, Wells DA, Lovell GJ, Ogle CK. 2002. Thermal injury-induced increases of hepatocyte SOCS3 lead to decreases in STAT3. Shock 18: 374379.
  • Krebs DL, Hilton DJ. 2001. SOCS proteins: negative regulators of cytokine signaling. Stem Cells 19: 378387.
  • LaBonne C, Whitman M. 1997. Localization of MAP kinase activity in early Xenopus embryos: implications for endogenous FGF signaling. Dev Biol 183: 920.
  • Marine JC, McKay C, Wang D, Topham DJ, Parganas E, Nakajima H, Pendeville H, Yasukawa H, Sasaki A, Yoshimura A, Ihle JN. 1999. SOCS3 is essential in the regulation of fetal liver erythropoiesis. Cell 98: 617627.
  • Nieuwkoop PD, Faber J. 1994. Normal table of Xenopus laevis (Daudin): a systematic and chronological survey of the development from the fertilized egg till the end of metamorphosis. New York, London: Garland Publishing, Inc. 252 p.
  • Ogle CK, Kong F, Guo X, Wells DA, Aosasa S, Noel G, Horseman N. 2000. The effect of burn injury on suppressors of cytokine signalling. Shock 14: 392398; discussion 398–399.
  • O'Shea JJ, Gadina M, Schreiber RD. 2002. Cytokine signaling in 2002: new surprises in the Jak/Stat pathway. Cell 109(Suppl): S121S131.
  • Roberts AW, Robb L, Rakar S, Hartley L, Cluse L, Nicola NA, Metcalf D, Hilton DJ, Alexander WS. 2001. Placental defects and embryonic lethality in mice lacking suppressor of cytokine signaling 3. Proc Natl Acad Sci U S A 98: 93249329.
  • Sive HA, Grainger RM, Harland RM. 1998. Early development of Xenopus laevis: a laboratory manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. 338 p.
  • Yasukawa H, Hoshijima M, Gu Y, Nakamura T, Pradervand S, Hanada T, Hanakawa Y, Yoshimura A, Ross J Jr, Chien KR. 2001. Suppressor of cytokine signaling-3 is a biomechanical stress-inducible gene that suppresses gp130-mediated cardiac myocyte hypertrophy and survival pathways. J Clin Invest 108: 14591467.