<i>Foxj1</i> as a target gene for RFX4.

<p>(A) qPCR. <i>Foxj1</i> mRNA levels were decreased in E12.5 KO brains, whereas there were no significant differences in <i>Ift172</i>, <i>Thbs</i>, <i>Dync2li1</i>, <i>Rfx2</i>, <i>Rfx3</i> mRNAs, and almost undetectable <i>Rfx4</i> transcripts compared to the controls (P< 0.0001, n = 5). (B) ChIP Assay. Fragmented DNA from adult brain was enriched by RFX4 antibody in the <i>Foxj1</i> promoter; the PCR bands shown were from flanking X-box1 and X-box2 primers (upper panel). In the <i>Dync2li1</i> and <i>Ift 172</i> promoter X-box regions, PCR bands were detected from samples immunoprecipitated with RFX4 antibodies (lower panel). Lane f/+: the PCR band was from control mouse DNA as a positive control; lane IgG: mouse IgG as a negative control; lane IP: RFX4 antibody was used to immunoprecipitate DNA. (C) Schematic diagram of the <i>Foxj1</i> X-box constructs, each representing where the putative X box is located on the genomic region. There are two putative X-boxes in the <i>Foxj1</i> proximal promoter region. The lowest illustrated construct contains no X-box and served as a negative control. (D) Luciferase reporter assay. The <i>Dync2li1</i> X-box sequence cloned into the luciferase reporter vector was <i>trans</i>-activated to the greatest extent by <i>Rfx4</i> cDNA overexpression in the cellular system (p< 0.0001), whereas there was relatively less <i>trans</i>-activation by X-boxes from <i>Foxj1</i> and <i>Ift172</i> promoters (p< 0.01). Fold changes were calculated by normalization to the expression of the pGL4.23 vector. The assay was carried out in triplicate, and the results were presented as fold change, with the values representing the means from three independent transfection experiments.</p

Conditional knockout and genotyping strategies.

<p>(A) Schematic version of the strategy used for the generation of the floxed <i>Rfx4</i> KO mice. In the WT allele, exon 4 was chosen for deletion. In the floxed allele, two loxP sites flanked exon 4, and a neomycin (neo) cassette with two Frt sites was inserted. Cre recombinase causes deletion of exon 4. “G1F”, on the 5’ arm, and “G1R”, on the 3’ arm, are genotyping PCR primers which produce WT, flox or KO bands. (B) Genotyping PCR: Lane +/- refers to a <i>Rfx4</i><b><i>^+/-</i></b> mouse, and the two bands were from floxed and KO alleles from a KO mouse; lane f/+ refers to a control mouse, and the two bands were from floxed and WT alleles from a <i>Rfx4</i><b><i>^f/+</i></b>mouse. (C) RT-PCR demonstrated a lower band in <i>Rfx4</i><b><i>^+/-</i></b> brain and spinal cord, representing the transcript produced by the KO allele. (D) As determined by qPCR, <i>Rfx4</i> mRNA levels were decreased by approximately 50% (P< 0.01, n = 4) in the brains of <i>Rfx4</i><b><i>^+/-</i></b> mice, as determined by fold changes after normalizing with <i>Gapdh</i> mRNA. (E) Image of the domed head of a mouse at 4 weeks of age (left) that was heterozygous for the <i>Rfx4</i> KO.</p

<i>Rfx4</i>^-/- embryos at E12.5 and E14.5 exhibit holoprosencephaly.

<p>Note the complete absence of the developing cerebral hemispheres (CH) in the KO embryo seen in A-C. There was a striking decrease in size of all regions of the brain, but especially the developing midbrain in (D). In addition, the developing mesencephalic aqueduct was absent, and marked neuroepithelial dysplasia was present in the caudal brainstem in (E), characterized by generalized disorganized growth and numerous rosettes. The 4^th ventricle was nearly absent in the KO embryo seen in (E-F). Scale bar = 20 μm. (G) The neuroepithelial cells of embryos at E 12.5 were labeled with the Ki67 antibody; the KO embryos (lower right panels) showed less staining overall. However, rosettes representing underdeveloped sites were extensively stained. Scale bar = 10 μm. (H) In representative staining of the brain sections with H&E at E14.5, there was a single ventricle without cerebral hemispheres in the forebrain (lower left panels). LV, lateral ventricle; BG, basal ganglia; Mb, midbrain; Th, thalamus; MA, mesencephalic aqueduct; 3^rd V, 3^rd ventricle; 4^th V, 4^th ventricle; BS, brain stem. Scale bar = 10 μm.</p

Characterization of <i>Rfx4^+/-</i> mice with hydrocephalus.

<p>(A) H&E staining. The left panels are representative slides from a control mouse at 4 weeks of age (<i>Rfx</i>^{<b><i>f/+</i></b>}), and the right panels were from a <i>Rfx4</i><b><i>^+/-</i></b> mouse, with roughly equivalent sections shown descending from rostral to caudal. The sections demonstrate the enlarged lateral and third ventricles, as indicated by the arrows, but not fourth ventricle enlargement, in the <i>Rfx4</i>^+/- mouse (representative of 6 <i>Rfx4</i>^{^+/-} mice and 4 controls). The middle right panel shows the emergence of false ventricles, and the white matter appeared to be compressed from the severe hydrocephalus. Scale bar = 20 μm. (B) SCO hypoplasia in <i>Rfx4</i>^+/- mice. The left panels of B showed the SCO stained with H&E, with a section from a control mouse (<i>Rfx</i>^<i>f/+</i>) on top, and from an <i>Rfx4</i>^+/- KO mouse on the bottom. Neighboring sections were stained with an antibody to Reissner’s fibers, and the resulting immunohistochemistry is shown on the right. Scale bar = 10 μm. (C) IHC quantitation. SCO length was measured by counting positively stained Reissner’s fibers slides from 3 controls and 3 <i>Rfx4</i>^+/- mice, using adjacent sections cut 5 μm apart. These measurements showed that SCO length in <i>Rfx4</i>^+/- KO mice was decreased when compared to controls (p<0.01). (D) Immunofluorescence staining. Acetylated alpha-tubulin labeled cilia (red fluorescence, blue is DAPI) were detached, patchy or missing from the walls of the ventricles in <i>Rfx4</i>^+/- mice (lower five panels). Scale bar = 5 μm.</p

Testing an Aflatoxin B1 Gene Signature in Rat Archival Tissues

Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c, and the C8orf46 homologue. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 μg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (<i>p</i> ≤ 0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and β-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25–500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in the liver, the primary target for AFB1-induced tumors. We conclude that an evaluation of gene signatures in archival tissues can be an important toxicological tool for evaluating critical molecular events associated with chemical exposures

Regeneration of injured epithelium after diacetyl instillation.

<p>a, b, c, d, Co-immunodetection of CCSP (red) and BrdU (green), with DAPI, a nuclear counter-stain (blue). a, An airway from a control rodent at day 3 shows expected distribution of CCSP with no BrdU detection (100x). b, An early lesion from a rodent treated with diacetyl (DA) shows significant epithelial regeneration detected by BrdU by day 3 (200x). c, A moderate BO lesion seen at day 3 after DA exposure shows significant BrdU staining along the epithelium as well as within the fibrotic lesion (arrow) (200x). As seen in b and c, regions of regeneration do not co-localize with epithelial CCSP staining. d, In an advanced BO lesion at day 7, CCSP distribution remains abnormal and diminished as compared to normal, and regeneration is also diminished (200x).</p

Injured epithelium regenerates, shows abnormal cellular composition, and progresses to BO.

<p>a, d, g, show co-immunodetection of CCSP (red) and acetylated tubulin (AcT) (green). b, e, h, are co-immunostained for CCSP (red), and SMA (green). c, f, i, are co-immunostained for CCSP (red) and β-catenin (green). DAPI, a nuclear counter-stain, is shown in blue. a, b, c, represent an airway from a control rodent (200x). d, e, f, represent an early lesion from a DA-treated rodent 3 days after instillation (200x). g, h, i represent an advanced lesion airway from a DA-treated rodent 7 days after instillation (400x). Three days after DA, the β-catenin expression appears normal, showing that epithelial repopulation has occurred (f), but CCSP and AcT expression is markedly diminished (d, e) demonstrating that the repopulated epithelium does not exhibit the normal airway cellular composition. Similar to day 3, the advanced day 7 BO lesion also shows a loss of CCSP and AcT expression; however, β-catenin expression is also irregular reflecting the distorted epithelium overlying the intraluminal polypoid lesions.</p

Severe epithelial injury with loss of normal epithelial structure occurs one day after Diacetyl treatment.

<p>a, d, Co-immunodetection of CCSP (red), and acetylated tubulin (AcT), (green). b, e, Co-immunostained for CCSP (red), and SMA (green). c, f, Co-immunostained for CCSP (red) and β-catenin (green). DAPI, a nuclear counter-stain, is shown in blue. Airways from control rodents (a,b,c) show baseline expression in controls of CCSP, AcT, SMA, and β-catenin (200x). CCSP and AcT (a) are expressed throughout a small airway, with a thin layer of SMA (b) surrounding the airway. The β-catenin staining shows membrane associated expression (c). Airways from DA-treated rodents one day following exposure (d, e, f) demonstrate severe depletion of epithelial protein expression of CCSP, AcT (d), and β-catenin (f), (400x). The β-catenin staining (f) also illustrates a flattened post injury cellular structure.</p

Neutrophil Influx into the Lung Fluid of Diacetyl-treated Rats.

a<p>5–9 animals/group.</p>b<p>Mean cell number (x10⁶) ± SD.</p><p>*Significantly different (p<0.05) from respective controls.</p

Diacetyl exposure results in early epithelial loss of CCSP transcript followed by collagen deposition.

<p>Whole lung transcript analysis using real-time PCR mRNA levels from rodents treated with water or diacetyl (DA). At each time-point (day 1, 3, and 7) DA-treated rodents were all compared to their appropriate water treated counterparts. a, CCSP transcript is significantly down-regulated at days 1,3, and 7. b, SPC transcript is minimally affected 1 day post DA treatment, and recovers to expression levels similar to water control at days 3 and 7. c, Collagen 1α-1 transcript is significantly up-regulated at days 3 and 7 post DA treatment, consistent with BO histology.</p