Pituitary <i>Mt1</i> expression is unaltered in adult <i>Egr-1⁻</i>^/− mice.

<p>Brain and pituitary tissue from adult wild type (WT) and <i>Egr-1⁻</i>^/− mice was dissected with the pituitary stalk intact, frozen on dry ice and stored at −80°C. Twenty micron sagittal sections were cut and <i>Mt1</i> mRNA expression determined by in situ hybridisation histochemistry. Quantification of <i>Mt1</i> expression by densitometry revealed no significant difference (p>0.05 unpaired t-test) of genotype. Representative autoradiographs are shown above the respective bar.</p

Treatment of rats with GnRH antagonist impairs reproductive status but does not affect <i>Mt1</i> expression.

<p>Male Wistar rats were given a daily i.p. injection of the GnRH antagonist cetrorelix (100 µg/day) or saline control for 4 weeks. (A) Serum LH was measured by ELISA. (B–C) Testis morphology was assessed by (B) paired testis weight and (C) histological analysis of tissue sections using hematoxylin and eosin staining. Scale bar = 25 µm (D–E) Brain and pituitary tissue from saline and cetrorelix-treated rats was dissected with the pituitary stalk intact, frozen on dry ice and stored at −80°C. Twenty micron sagittal sections were cut and <i>Mt1</i> mRNA expression determined by in situ hybridisation histochemistry. (D) Representative autoradiographs. In both treatment groups, strong pituitary expression was observed in the pars tuberalis and along the rostral extent of the ventral pars distalis; weaker expression was observed throughout the rest of the pars distalis. (E) Quantification of <i>Mt1</i> expression by densitometry. *** p<0.001 saline vs cetrorelix group (unpaired t-test). Sal: saline-treated; Cet: cetrorelix-treated.</p

Regulation of rat <i>Mt1</i> promoter activity by PITX-1 and EGR-1 in vitro.

<p>COS-7 cells were co-transfected with an <i>Mt1</i>-luciferase reporter construct and a combination of control vector, PITX-1 expression vector and EGR-1 expression vector. Horizontal bars indicate mutagenesis of PITX-1 and EGR-1 binding sites. P1: distal PITX-1 consensus site; P2: proximal PITX-1 consensus site; E: EGR-1 consensus site. *** p<0.001 vs control group; ### p<0.001 vs PITX-1 group (one-way ANOVA with Bonferroni post-hoc test).</p

GnRH agonist treatment induces EGR-1 and inhibits <i>Mt1</i> expression in αT3-1 gonadotroph cells.

<p>Cells were treated with 100(A) Total mRNA was extracted for analysis of <i>Egr-1</i> mRNA, expressed relative to <i>Gapdh</i>, by qRT-PCR. *** p<0.001 vs time 0 (one-way ANOVA with Bonferroni post-hoc test). (B) Cytoplasmic and nuclear-enriched lysates were prepared for analysis of EGR-1 and actin protein expression by western blot. (C) Following 20 hours of GnRH agonist treatment, total mRNA was extracted for analysis of <i>Mt1</i> mRNA, expressed relative to <i>Gapdh</i>, by qRT-PCR. * p<0.05 control vs treated group (unpaired t-test).</p

Similarity and identity analysis of CD4-related genes between mammals and teleosts.

<p>Similarity and identity analysis of CD4-related genes between mammals and teleosts.</p

Zebrafish CD4-1/CD4-2 gene distribution.

<p>(A) Tissue/blood expression of CD4-1/CD4-2 genes in zebrafish. (B) Cell profile obtained from FACS sorted zf kidney and spleen cells. E = erythrocytes, L = lymphocytes, P = (hemopoietic) precursor cells and M/G = monocytes/granulocytes. (C) Differential expression of the zfCD4-1/CD4-2 genes within the FACS sorted populations. *, p<0.05.</p

The transcript levels of different cytokines in CD4-1⁺ sorted lymphocytes from kidney and spleen of zebrafish given antigen for the first time, or following boosting with specific or non-specific antigen, were determined by QRT-PCR.

<p>Data were first normalized to the expression level of EF-1α, and then expressed as a fold change by comparing the transcript levels in samples from fish primed and boosted with HGG (HGG:HGG), in comparison to fish initially given PBS followed by antigen for the first time (e.g. PBS:HGG, or PBS:OVA), or fish given HGG initially followed by OVA (HGG:OVA). The values presented are means of 4 replicates (of 5 pooled fish). *, P<0.05 between the two groups compared in each graph.</p

The transcript levels of IFN-γ and IL-4/13B in kidney of zebrafish given antigen for the first time, or following boosting with specific or non-specific antigen, were determined by QRT-PCR.

<p>Each bar represents the expression relative to EF-1α. PBS then OVA: fish given PBS and subsequently primed with OVA. PBS then HGG: fish given PBS and subsequently primed with HGG. HGG then OVA: fish primed with HGG and subsequently boosted with OVA. HGG then HGG: fish primed with HGG and subsequently boosted with HGG. The mean values are presented for 6 individual fish. *, significantly different (P<0.05) against PBS injected fish.</p

CD4-1 promoter analysis in zebrafish.

<p>In silico analysis of putative promoter and the first 1kb of the first intron of zfCD4-1. Transcription factor binding sites are shown. The sequence orientation of the transcription factor binding sites is given as upper (+ strand) and lower (- strand) annotation. cMyb = a member of the myeloblastosis family; GATA = GATA- binding factor; IRF = interferon regulatory factor family members; RUNX = Runt-related transcription factor; STAT = signal transducer and activator of transcription; TATA = cellular and viral TATA box elements. Exon 1 is coloured black.</p

Detection of zfCD4-1⁺ cells using immunofluorescence assay and immuohistochemical assays.

<p>(A) Immunofluorescence staining of the zfCD4-1 molecule on zebrafish leukocytes. The cells stained with DAPI (blue) for counterstaining nuclei (left panel), the zfCD4-1 polyclonal followed by FITC labelled (green) secondary antibody (middle panel) and merged image (right panel) are shown. Scale bar = 10 μm). The arrow shows a cell expressing CD4 on its surface. (B) Cells from (A) were also visualized using a confocal microscope. (C) Double immunofluorescence staining of zebrafish peripheral lymphocytes in gut sections incubated with rabbit anti zfCD4-1 and mouse anti human ZAP70. ZAP70^<b>+</b> cells are green and CD4^<b>+</b> cells are red. Co-localization was confirmed by a Z-stack image analysis using Zeiss confocal microscopy. The data are representative results obtained from three independent experiments. (D) CD4-1^<b>+</b> cells present within the cuff of leukocytes surrounding the granulomas developed after <i>M</i>. <i>marinum</i> EspG5::Tn mutant infection of zebrafish 4 weeks earlier. Top images 200x, bottom images 400x. Left hand images used anti-CD4-1 serum, right hand images are controls.</p