The HIV-1 envelope protein gp120 impairs B cell proliferation by inducing TGF-ß1 production and FcRL4 expression.

The humoral immune response after acute infection with HIV-1 is delayed and ineffective. The HIV-1 envelope protein gp120 binds to and signals through integrin α4ß7 on T cells. We found that gp120 also bound to and signaled through α4ß7 on naive B cells, which resulted in an abortive proliferative response. In primary B cells, signaling by gp120 through α4ß7 resulted in increased expression of the immunosuppressive cytokine TGF-ß1 and FcRL4, an inhibitory receptor expressed on B cells. Coculture of B cells with HIV-1-infected autologous CD4(+) T cells also increased the expression of FcRL4 by B cells. Our findings indicated that in addition to mediating chronic activation of the immune system, viral proteins contributed directly to HIV-1-associated B cell dysfunction. Our studies identify a mechanism whereby the virus may subvert the early HIV-1-specific humoral immune response.

HIV-1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells.

Infection with human immunodeficiency virus 1 (HIV-1) results in the dissemination of virus to gut-associated lymphoid tissue. Subsequently, HIV-1 mediates massive depletion of gut CD4+ T cells, which contributes to HIV-1-induced immune dysfunction. The migration of lymphocytes to gut-associated lymphoid tissue is mediated by integrin alpha4beta7. We demonstrate here that the HIV-1 envelope protein gp120 bound to an activated form of alpha4beta7. This interaction was mediated by a tripeptide in the V2 loop of gp120, a peptide motif that mimics structures presented by the natural ligands of alpha4beta7. On CD4+ T cells, engagement of alpha4beta7 by gp120 resulted in rapid activation of LFA-1, the central integrin involved in the establishment of virological synapses, which facilitate efficient cell-to-cell spreading of HIV-1.

Interleukin-2 inhibits HIV-1 replication in some human T cell lymphotrophic virus-1-infected cell lines via the induction and incorporation of APOBEC3G into the virion.

IL-2 has been used in culture of primary T cells to maintain cell proliferation. We have previously reported that IL-27 inhibits HIV-1 replication in primary T cells in the presence of IL-2. To gain a better understanding of the mechanisms involved in this inhibitory effect, we attempted to investigate in detail the effects of IL-27 and IL-2 using several cell lines. Unexpectedly, IL-27 did not inhibit HIV-1 in T cell lines, whereas IL-2 inhibited HIV-1 replication in the human T cell lymphotrophic virus (HTLV)-1-transformed T cell lines, MT-2, MT-4, SLB-1, and ATL-2. No effects were seen in HTLV-1-negative cell lines. Utilizing MT-2 cells, we demonstrated that IL-2 treatment inhibited HIV-1 syncytia-inducing ability and dose-dependently decreased supernatant p24 antigen levels by >90%. Using real time PCR and Western blot analysis, we observed that IL-2 treatment induced the host restriction factor, APOBEC3G with accumulation into the lower molecular mass active form as characterized by FPLC. Further analysis revealed that the virus recovered from IL-2-treated MT-2 cells had impaired replication competency. This was found to be due to incorporation of APOBEC3G into the virion despite the presence of Vif. These findings demonstrate a novel role for IL-2 in regulating production of infectious HIV-1 virions in HTLV-1-infected cells through the induction of APOBEC3G.

Interferon stimulated exonuclease gene 20 kDa links psychiatric events to distinct hepatitis C virus responses in human immunodeficiency virus positive patients.

Hepatitis C Virus (HCV) infection occurs frequently in patients with preexisting mental illness. Treatment for chronic hepatitis C using interferon formulations often increases risk for neuro-psychiatric symptoms. Pegylated-Interferon-α (PegIFN-α) remains crucial for attaining sustained virologic response (SVR); however, PegIFN-α based treatment is associated with psychiatric adverse effects, which require dose reduction and/or interruption. This study's main objective was to identify genes induced by PegIFN-α and expressed in the central nervous system and immune system, which could mediate the development of psychiatric toxicity in association with antiviral outcome. Using peripheral blood mononuclear cells from Human Immunodeficiency Virus (HIV)/HCV co-infected donors (N = 28), DNA microarray analysis was performed and 21 differentially regulated genes were identified in patients with psychiatric toxicity versus those without. Using these 21 expression profiles a two-way-ANOVA was performed to select genes based on antiviral outcome and occurrence of neuro-psychiatric adverse events. Microarray analysis demonstrated that Interferon-stimulated-exonuclease-gene 20 kDa (ISG20) and Interferon-alpha-inducible-protein 27 (IFI27) were the most regulated genes (P < 0.05) between three groups that were built by combining antiviral outcome and neuro-psychiatric toxicity. Validation by bDNA assay confirmed that ISG20 expression levels were significantly associated with these outcomes (P < 0.035). Baseline levels and induction of ISG20 correlated independently with no occurrence of psychiatric adverse events and non-response to therapy (P < 0.001). Among the 21 genes that were associated with psychiatric adverse events and 20 Interferon-inducible genes (IFIGs) used as controls, only ISG20 expression was able to link PegIFN-α related neuro-psychiatric toxicity to distinct HCV-responses in patients co-infected with HIV and HCV in vivo.

Sequencing and characterization of the complete mitochondrial genomes of three Pneumocystis species provide new insights into divergence between human and rodent Pneumocystis.

Pneumocystis jirovecii is an important opportunistic pathogen associated with AIDS and other immunodeficient conditions. Currently, very little is known about its nuclear and mitochondrial genomes. In this study, we sequenced the complete mitochondrial genome (mtDNA) of this organism and its closely related species Pneumocystis carinii and Pneumocystis murina by a combination of sequencing technologies. Our study shows that P. carinii and P. murina mtDNA share a nearly identical number and order of genes in a linear configuration, whereas P. jirovecii has a circular mtDNA containing nearly the same set of genes but in a different order. Detailed studies of the mtDNA terminal structures of P. murina and P. carinii suggest a unique replication mechanism for linear mtDNA. Phylogenetic analysis supports a close association of Pneumocystis species with Taphrina, Saitoella, and Schizosaccharomyces, and divergence within Pneumocystis species, with P. murina and P. carinii being more closely related to each other than either is to P. jirovecii. Comparative analysis of four complete P. jirovecii mtDNA sequences in this study and previously reported mtDNA sequences for diagnosing and genotyping suggests that the current diagnostic and typing methods can be improved using the complete mtDNA data. The availability of the complete P. jirovecii mtDNA also opens the possibility of identifying new therapeutic targets.

The CD8+ HLA-DR+ T cells expanded in HIV-1 infection are qualitatively identical to those from healthy controls.

HIV-induced immune activation leads to expansion of a subset of human CD8(+) T cells expressing HLA-DR antigens. Expansion of CD8(+) HLA-DR(+) T cells can be also observed in non-HIV settings including several autoimmune diseases and aging. Although these cells are felt to represent "immune exhaustion" and/or to be anergic, their precise role in host defense has remained unclear. Here, we report that this subset of cells exhibits a restricted repertoire, shows evidence of multiple rounds of division, but lacks markers of recent TCR engagement. Detailed cell cycle analysis revealed that compared with their CD8(+) HLA-DR(-) counterpart, the CD8(+) HLA-DR(+) T-cell pool contained an increased fraction of cells in S-phase with elevated levels of the G2/M regulators: cyclin A2, CDC25C, Cdc2 (CDK1), indicating that these cells are not truly anergic but rather experiencing proliferation in vivo. Together, these data support a hypothesis that antigen stimulation leads to the initial expansion of a CD8(+) pool of cells in vivo that undergo further expansion independent of ongoing TCR engagement. No qualitative differences were noted between CD8(+) HLA-DR(+) cells from HIV(+) and HIV(-) donors, indicating that the generation of CD8(+) HLA-DR(+) T cells is a part of normal immune regulation that is exaggerated in the setting of HIV-1 infection.

Interleukin-27 treated human macrophages induce the expression of novel microRNAs which may mediate anti-viral properties.

Interleukin-27 (IL-27) is a pleiotropic cytokine which plays important and diverse roles in the immune system. We have previously demonstrated that IL-27 induces potent anti-viral effects against HIV-1, HIV-2, SIV, HSV-2, KSHV and influenza viruses in macrophages. This induction occurred in an interferon (IFN) independent manner and involved down regulation of SPTBN1. MicroRNAs (miRNAs) are critical regulators of mRNA translation and turnover. There have been reports that some miRNAs inhibit viral replication. In this study, we hypothesized that IL-27 could induce the expression of novel miRNAs in macrophages which may have functional relevance in terms of anti-viral activity and primary monocytes were differentiated into macrophages using either M-CSF (M-Mac) or a combination of M-CSF and IL-27 (I-Mac) for seven days. Following this, total RNA was extracted from these cells and deep sequencing was performed, in parallel with gene expression microarrays. Using the novel miRNA discovery software, miRDeep, seven novel miRNAs were discovered in these macrophages. Four of which were preferentially expressed in I-Mac (miR-SX1, -SX2, -SX3 and -SX6) whilst three were detected in both M-Mac and I-Mac (miR-SX4, -SX5 and -SX7). The expression of six of the seven novel miRNAs was highly correlated with qRT-PCR using specific primer/probes designed for the novel miRNAs. Gene expression microarray further demonstrated that a number of genes were potentially targeted by these differentially expressed novel miRNAs. Finally, several of these novel miRNAs (miR-SX1, -SX4, -SX5, -SX6 and -SX7) were shown to target the open reading frames of a number of viruses (including HSV-1, HSV-2 and HHV-8) which may partially explain the anti-viral properties observed.

DAVID-WS: a stateful web service to facilitate gene/protein list analysis.

SUMMARY: The database for annotation, visualization and integrated discovery (DAVID), which can be freely accessed at http://david.abcc.ncifcrf.gov/, is a web-based online bioinformatics resource that aims to provide tools for the functional interpretation of large lists of genes/proteins. It has been used by researchers from more than 5000 institutes worldwide, with a daily submission rate of ∼1200 gene lists from ∼400 unique researchers, and has been cited by more than 6000 scientific publications. However, the current web interface does not support programmatic access to DAVID, and the uniform resource locator (URL)-based application programming interface (API) has a limit on URL size and is stateless in nature as it uses URL request and response messages to communicate with the server, without keeping any state-related details. DAVID-WS (web service) has been developed to automate user tasks by providing stateful web services to access DAVID programmatically without the need for human interactions. AVAILABILITY: The web service and sample clients (written in Java, Perl, Python and Matlab) are made freely available under the DAVID License at http://david.abcc.ncifcrf.gov/content.jsp?file=WS.html.

High interferon-stimulated gene ISG-15 expression affects HCV treatment outcome in patients co-infected with HIV and HCV.

Increased baseline expression and lack of induction of interferon-stimulated genes (ISG) are strong negative predictors of therapeutic response to PegIFN/RBV in patients co-infected with HIV and hepatitis C virus (HCV). This study specifically addressed whether ISG-15 expression influences therapeutic responses in 20 HIV/HCV genotype-1 subjects undergoing HCV treatment. Non-responders had significantly higher baseline expression and selective induction of ISG-15 after IFN-α treatment relative to participants with sustained virological response. High baseline levels of ISG-15 were also associated with less induction of ISG with treatment. These results support a role for ISG-15 as a prognostic indicator and resistance factor to IFN-α.

Differential effects of HIV viral load and CD4 count on proliferation of naive and memory CD4 and CD8 T lymphocytes.

We previously showed that HIV infection leads to expansion of a rapidly proliferating pool (s(1)) of CD4 and CD8 T lymphocytes. In the current study, we used in vivo labeling with bromodeoxyuridine to characterize the kinetics of naive, memory, and activated (HLA-DR(+)/CD38(+)) subpopulations of CD4 and CD8 T lymphocytes, and to examine the relationship between kinetic parameters and baseline CD4 counts, HIV viral load, potential markers of microbial translocation, and cytokine levels. Activated cells showed the highest proliferation rates, followed by effector and central memory cells, with naive cells showing the lowest rates, for both CD4 and CD8 T cells. HIV viral load correlated with s(1) of CD4 and CD8 effector memory cells, as well as CD8 naive cells, whereas CD4 cell counts correlated inversely with naive CD4 s(1). Endotoxin levels showed a weak negative association with CD4 but not CD8 s(1). INF-γ and TNF-α were associated with s(1) for CD4 and CD8 cells, respectively. Thus, HIV is the primary driving force behind the activation and proliferation of most subsets of both CD4 and CD8 T lymphocytes, whereas naive CD4 cell proliferation likely represents a homeostatic response. Microbial translocation does not appear to play an important role in this proliferation.

Cutting edge: Ku70 is a novel cytosolic DNA sensor that induces type III rather than type I IFN.

Cytosolic foreign DNA is detected by pattern recognition receptors and mainly induces type I IFN production. We found that transfection of different types of DNA into various untreated cells induces type III IFN (IFN-λ1) rather than type I IFN, indicating the presence of uncharacterized DNA sensor(s). A pull-down assay using cytosolic proteins identified that Ku70 and Ku80 are the DNA-binding proteins. The knockdown studies and the reporter assay revealed that Ku70 is a novel DNA sensor inducing the IFN-lambda1 activation. The functional analysis of IFNL1 promoter revealed that positive-regulatory domain I and IFN-stimulated response element sites are predominantly involved in the DNA-mediated IFNL1 activation. A pull-down assay using nuclear proteins demonstrated that the IFN-λ1 induction is associated with the activation of IFN regulatory factor-1 and -7. Thus, to our knowledge, we show for the first time that Ku70 mediates type III IFN induction by DNA.

Evolution of the pygmy phenotype: evidence of positive selection fro genome-wide scans in African, Asian, and Melanesian pygmies.

Human pygmy populations inhabit different regions of the world, from Africa to Melanesia. In Asia, short-statured populations are often referred to as "negritos." Their short stature has been interpreted as a consequence of thermoregulatory, nutritional, and/or locomotory adaptations to life in tropical forests. A more recent hypothesis proposes that their stature is the outcome of a life history trade-off in high-mortality environments, where early reproduction is favored and, consequently, early sexual maturation and early growth cessation have coevolved. Some serological evidence of deficiencies in the growth hormone/insulin-like growth factor axis have been previously associated with pygmies' short stature. Using genome-wide single-nucleotide polymorphism genotype data, we first tested whether different negrito groups living in the Philippines and Papua New Guinea are closely related and then investigated genomic signals of recent positive selection in African, Asian, and Papuan pygmy populations. We found that negritos in the Philippines and Papua New Guinea are genetically more similar to their nonpygmy neighbors than to one another and have experienced positive selection at different genes. These results indicate that geographically distant pygmy groups are likely to have evolved their short stature independently. We also found that selection on common height variants is unlikely to explain their short stature and that different genes associated with growth, thyroid function, and sexual development are under selection in different pygmy groups.

Host gene expression changes correlating with anti-HIV-1 effects in human subjects after treatment with peginterferon Alfa-2a.

We investigated whether interferon-inducible genes (IFIGs) with known anti-human immunodeficiency virus (HIV) activity in vitro were associated with in vivo virological response in HIV infection. Nine untreated HIV-1-infected volunteers were treated for 12 weeks with peginterferon alfa-2a. A subset of IFIGs (23 of 47) increased compared with baseline through 6 weeks beyond therapy, and 10 of the 23 IFIGs significantly inversely correlated (r = -0.7; P < .05) with virological response. The strength of peginterferon alfa-2a-induced IFIG response significantly correlated with declines in HIV load during treatment (r(2) = 0.87, p = .003). This study links HIV virological response to a specific IFIG subset, a potential prognostic indicator in peginterferon alfa-2a-treated patients with HIV infection.

HIV infection and antiretroviral therapy have divergent effects on mitochondria in adipose tissue.

BACKGROUND: Although human immunodeficiency virus (HIV) infection and antiretroviral therapy (ART) affect mitochondrial DNA (mtDNA) content and function, comprehensive evaluations of their effects on mitochondria in muscle, adipose tissue, and blood cells are limited. METHODS: Mitochondrial DNA quantification, mitochondrial genome sequencing, and gene expression analysis were performed on muscle, adipose tissue, and peripheral blood mononuclear cell (PBMC) samples from untreated HIV-positive patients, HIV-positive patients receiving nucleoside reverse transcriptase inhibitor (NRTI)-based ART, and HIV-negative controls. RESULTS: The adipose tissue mtDNA/nuclear DNA (nDNA) ratio was increased in untreated HIV-infected patients (ratio, 353) and decreased in those receiving ART (ratio, 162) compared with controls (ratio, 255; P < .05 for both comparisons); the difference between the 2 HIV-infected groups was also significant (P = .002). In HIV-infected participants, mtDNA/nDNA in adipose tissue correlated with the level of activation (CD38+ /HLA-DR+) for CD4+ and CD8+ lymphocytes. No significant differences in mtDNA content were noted in muscle or PMBCs among groups. Exploratory DNA microarray analysis identified differential gene expression between patient groups, including a subset of adipose tissue genes. CONCLUSIONS: HIV infection and ART have opposing effects on mtDNA content in adipose tissue; immune activation may mediate the effects of HIV, whereas NRTIs likely mediate the effects of ART.

Altered expression of SHIP, a Toll-like receptor pathway inhibitor, is associated with the severity of liver fibrosis in chronic hepatitis C virus infection.

Hepatitis C-related fibrogenesis has been shown to involve complex interactions between peripheral and hepatic immune responses. Peripheral whole blood (PB) samples from patients with chronic hepatitis C (n = 36) were subjected to microarray analysis in order to identify gene expression patterns associated with immune pathways in PB and hepatic fibrosis. Distinct regulation of gene expression of inositol polyphosphate-5-phosphatase/145kDa (INPP5D or SHIP), a TLR2/TLR4-inhibitor, and heat shock protein 8/22 kDa (HSPB8), an endogenous TLR4-ligand, during fibrogenesis was identified and could be confirmed by quantitative reverse-transcription polymerase chain reaction. These results suggest a potential link between peripheral activity of the TLR4-pathway, peripheral SHIP-dependent immune regulation, and liver fibrosis.

LIM kinase 1 - dependent cofilin 1 pathway and actin dynamics mediate nuclear retinoid receptor function in T lymphocytes.

BACKGROUND: It is known that retinoid receptor function is attenuated during T cell activation, a phenomenon that involves actin remodeling, suggesting that actin modification may play a role in such inhibition. Here we have investigated the role of actin dynamics and the effect of actin cytoskeleton modifying agents on retinoid receptor-mediated transactivation. RESULTS: Agents that disturb the F-actin assembly or disassembly attenuated receptor-mediated transcription indicating that actin cytoskeletal homeostasis is important for retinoid receptor function. Overexpression or siRNA-induced knockdown of cofilin-1 (CFL1), a key regulator of F-actin assembly, induced the loss of receptor function. In addition, expression of either constitutively active or inactive/dominant-negative mutants of CFL1or CFL1 kinase LIMK1 induced loss of receptor function suggesting a critical role of the LIMK1-mediated CFL1 pathway in receptor-dependent transcription. Further evidence of the role of LMK1/CFL1-mediated actin dynamics, was provided by studying the effect of Nef, an actin modifying HIV-1 protein, on receptor function. Expression of Nef induced phosphorylation of CFL1 at serine 3 and LIMK1 at threonine 508, inhibited retinoid-receptor mediated reporter activity, and the expression of a number of genes that contain retinoid receptor binding sites in their promoters. The results suggest that the Nef-mediated inhibition of receptor function encompasses deregulation of actin filament dynamics by LIMK1 activation and phosphorylation of CFL1. CONCLUSION: We have identified a critical role of LIMK1-mediated CFL1 pathway and actin dynamics in modulating retinoid receptor mediated function and shown that LIMK1-mediated phosphocycling of CFL1 plays a crucial role in maintaining actin homeostasis and receptor activity. We suggest that T cell activation-induced repression of nuclear receptor-dependent transactivation is in part through the modification of actin dynamics.

Gene expression patterns induced by HPV-16 L1 virus-like particles in leukocytes from vaccine recipients.

Human papillomavirus (HPV) virus-like particle (VLP) vaccines were recently licensed. Although neutralizing Ab titers are thought to be the main effectors of protection against infection, early predictors of long-term efficacy are not yet defined and a comprehensive understanding of innate and adaptive immune responses to vaccination is still lacking. Here, microarrays were used to compare the gene expression signature in HPV-16 L1 VLP-stimulated PBMCs from 17 vaccine and 4 placebo recipients before vaccination and 1 mo after receiving the second immunization. Vaccination with a monovalent HPV-16 L1 VLP vaccine was associated with modulation of genes involved in the inflammatory/defense response, cytokine, IFN, and cell cycle pathways in VLP-stimulated PBMCs. Additionally, there was up-regulation of probesets associated with cytotoxic (GZMB, TNFSF10) and regulatory (INDO, CTLA4) activities. The strongest correlations with neutralizing Ab titers were found for cyclin D2 (CCND2) and galectin (LGALS2). Twenty-two differentially expressed probesets were selected for confirmation by RT-PCR in an independent sample set. Agreement with microarray data was seen for more than two-thirds of these probesets. Up-regulation of immune/defense response genes by HPV-16 L1 VLP, in particular, IFN-induced genes, was observed in PBMCs collected before vaccination, with many of these genes being further induced following vaccination. In conclusion, we identified important innate and adaptive response-related genes induced by vaccination with HPV-16 L1 VLP. Further studies are needed to identify gene expression signatures of immunogenicity and long-term protection with potential utility in prediction of long-term HPV vaccination outcomes in clinical trials.

Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.

Functional analysis of large gene lists, derived in most cases from emerging high-throughput genomic, proteomic and bioinformatics scanning approaches, is still a challenging and daunting task. The gene-annotation enrichment analysis is a promising high-throughput strategy that increases the likelihood for investigators to identify biological processes most pertinent to their study. Approximately 68 bioinformatics enrichment tools that are currently available in the community are collected in this survey. Tools are uniquely categorized into three major classes, according to their underlying enrichment algorithms. The comprehensive collections, unique tool classifications and associated questions/issues will provide a more comprehensive and up-to-date view regarding the advantages, pitfalls and recent trends in a simpler tool-class level rather than by a tool-by-tool approach. Thus, the survey will help tool designers/developers and experienced end users understand the underlying algorithms and pertinent details of particular tool categories/tools, enabling them to make the best choices for their particular research interests.

Safety, tolerability, and mechanisms of antiretroviral activity of pegylated interferon Alfa-2a in HIV-1-monoinfected participants: a phase II clinical trial.

BACKGROUND: To our knowledge, the antiviral activity of pegylated interferon alfa-2a has not been studied in participants with untreated human immunodeficiency virus type 1 (HIV-1) infection but without chronic hepatitis C virus (HCV) infection. METHODS: Untreated HIV-1-infected volunteers without HCV infection received 180 microg of pegylated interferon alfa-2a weekly for 12 weeks. Changes in plasma HIV-1 RNA load, CD4(+) T cell counts, pharmacokinetics, pharmacodynamic measurements of 2',5'-oligoadenylate synthetase (OAS) activity, and induction levels of interferon-inducible genes (IFIGs) were measured. Nonparametric statistical analysis was performed. RESULTS: Eleven participants completed 12 weeks of therapy. The median plasma viral load decrease and change in CD4(+) T cell counts at week 12 were 0.61 log(10) copies/mL (90% confidence interval [CI], 0.20-1.18 log(10) copies/mL) and -44 cells/microL (90% CI, -95 to 85 cells/microL), respectively. There was no correlation between plasma viral load decreases and concurrent pegylated interferon plasma concentrations. However, participants with larger increases in OAS level exhibited greater decreases in plasma viral load at weeks 1 and 2 (r = -0.75 [90% CI, -0.93 to -0.28] and r = -0.61 [90% CI, -0.87 to -0.09], respectively; estimated Spearman rank correlation). Participants with higher baseline IFIG levels had smaller week 12 decreases in plasma viral load (0.66 log(10) copies/mL [90% CI, 0.06-0.91 log(10) copies/mL]), whereas those with larger IFIG induction levels exhibited larger decreases in plasma viral load (-0.74 log(10) copies/mL [90% CI, -0.93 to -0.21 log(10) copies/mL]). CONCLUSION: Pegylated interferon alfa-2a was well tolerated and exhibited statistically significant anti-HIV-1 activity in HIV-1-monoinfected patients. The anti-HIV-1 effect correlated with OAS protein levels (weeks 1 and 2) and IFIG induction levels (week 12) but not with pegylated interferon concentrations.

Pre-gastrula expression of zebrafish extraembryonic genes.

BACKGROUND: Many species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL). RESULTS: We hypothesize that key components of the yolk syncytial layer's mesoderm and endoderm inducing activity are expressed as mRNAs in the E-YSL. To identify genes expressed in the E-YSL, we used microarrays to compare the transcription profiles of intact pre-gastrula embryos with pre-gastrula embryonic cells that we had separated from the yolk and yolk syncytial layer. This identified a cohort of genes with enriched expression in intact embryos. Here we describe our whole mount in situ hybridization analysis of sixty-eight of them. This includes ten genes with E-YSL expression (camsap1l1, gata3, znf503, hnf1ba, slc26a1, slc40a1, gata6, gpr137bb, otop1 and cebpa), four genes with expression in the enveloping layer (EVL), a superficial epithelium that protects the embryo (zgc:136817, zgc:152778, slc14a2 and elovl6l), three EVL genes whose expression is transiently confined to the animal pole (elovl6l, zgc:136359 and clica), and six genes with transient maternal expression (mtf1, wu:fj59f04, mospd2, rftn2, arrdc1a and pho). We also assessed the requirement of Nodal signaling for the expression of selected genes in the E-YSL, EVL and margin. Margin expression was Nodal dependent for all genes we tested, including the concentrated margin expression of an EVL gene: zgc:110712. All other instances of EVL and E-YSL expression that we tested were Nodal independent. CONCLUSION: We have devised an effective strategy for enriching and identifying genes expressed in the E-YSL of pre-gastrula embryos. To our surprise, maternal genes and genes expressed in the EVL were also enriched by this strategy. A number of these genes are promising candidates for future functional studies on early embryonic patterning.