Endocardial Brg1 disruption illustrates the developmental origins of semilunar valve disease

AbstractThe formation of intricately organized aortic and pulmonic valves from primitive endocardial cushions of the outflow tract is a remarkable accomplishment of embryonic development. While not always initially pathologic, developmental semilunar valve (SLV) defects, including bicuspid aortic valve, frequently progress to a disease state in adults requiring valve replacement surgery. Disrupted embryonic growth, differentiation, and patterning events that “trigger” SLV disease are coordinated by gene expression changes in endocardial, myocardial, and cushion mesenchymal cells. We explored roles of chromatin regulation in valve gene regulatory networks by conditional inactivation of the Brg1-associated factor (BAF) chromatin remodeling complex in the endocardial lineage. Endocardial Brg1-deficient mouse embryos develop thickened and disorganized SLV cusps that frequently become bicuspid and myxomatous, including in surviving adults. These SLV disease-like phenotypes originate from deficient endocardial-to-mesenchymal transformation (EMT) in the proximal outflow tract (pOFT) cushions. The missing cells are replaced by compensating neural crest or other non-EMT-derived mesenchyme. However, these cells are incompetent to fully pattern the valve interstitium into distinct regions with specialized extracellular matrices. Transcriptomics reveal genes that may promote growth and patterning of SLVs and/or serve as disease-state biomarkers. Mechanistic studies of SLV disease genes should distinguish between disease origins and progression; the latter may reflect secondary responses to a disrupted developmental system

Rescue of Degradation-Prone Mutants of the FK506-Rapamycin Binding (FRB) Protein with Chemical Ligands

We recently reported that certain mutations in the FK506-rapamycin binding (FRB) domain disrupt its stability in vitro and in vivo (Stankunas et al. Mol. Cell , 2003 , 12 , 1615). To determine the precise residues that cause instability, we calculated the folding free energy (Δ G ) of a collection of FRB mutants by measuring their intrinsic tryptophan fluorescence during reversible chaotropic denaturation. Our results implicate the T2098L point mutation as a key determinant of instability. Further, we found that some of the mutants in this collection were destabilised by up to 6 kcal mol −1 relative to the wild type. To investigate how these mutants behave in cells, we expressed firefly luciferase fused to FRB mutants in African green monkey kidney (COS) cell lines and mouse embryonic fibroblasts (MEFs). When unstable FRB mutants were used, we found that the protein levels and the luminescence intensities were low. However, addition of a chemical ligand for FRB, rapamycin, restored luciferase activity. Interestingly, we found a roughly linear relationship between the Δ G of the FRB mutants calculated in vitro and the relative chemical rescue in cells. Because rapamycin is capable of simultaneously binding both FRB and the chaperone, FK506-binding protein (FKBP), we next examined whether FKBP might contribute to the protection of FRB mutants. Using both in vitro experiments and a cell-based model, we found that FKBP stabilizes the mutants. These findings are consistent with recent models that suggest damage to intrinsic Δ G can be corrected by pharmacological chaperones. Further, these results provide a collection of conditionally stable fusion partners for use in controlling protein stability.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56088/1/1162_ftp.pd

Transcriptomes of post-mitotic neurons identify the usage of alternative pathways during adult and embryonic neuronal differentiation

Original Panther analysis tool data tables showing values and p -values for overrepresentation data sets. (XLSX 21 kb

Spatial and Temporal Control of Transgene Expression in Zebrafish

<div><p>Transgenic zebrafish research has provided valuable insights into gene functions and cell behaviors directing vertebrate development, physiology, and disease models. Most approaches use constitutive transgene expression and therefore do not provide control over the timing or levels of transgene induction. We describe an inducible gene expression system that uses new tissue-specific zebrafish transgenic lines that express the Gal4 transcription factor fused to the estrogen-binding domain of the human estrogen receptor. We show these Gal4-ERT driver lines confer rapid, tissue-specific induction of UAS-controlled transgenes following tamoxifen exposure in both embryos and adult fish. We demonstrate how this technology can be used to define developmental windows of gene function by spatiotemporal-controlled expression of constitutively active Notch1 in embryos. Given the array of existing UAS lines, the modular nature of this system will enable many previously intractable zebrafish experiments.</p></div

Overexpression of the Notch1 intracellular domain using the <i>dusp6:Gal4-ERT</i> driver disrupts notochord development.

<p>(<b>A and B</b>) EGFP expression at the 10-somite stage in control (A) and 2 μM 4-OHT treated <i>Tg(dusp6:Gal4-ERT-VP16; UAS:EGFP)</i> fish. The green arrow points to EGFP expression at the midline. (<b>C–F</b>) DIC images of control (C and D) and 4-OHT treated (4 μM from 2–24 hpf, E and F) <i>Tg(dusp6:Gal4-ERT-VP16; UAS:NICD)</i> animals at 24 hpf. Regions bounded by the dashed red box in panels C and E are shown in high magnification in panels D and F, respectively. In panel D, the red arrow indicates the floor plate (fp) and the blue arrow indicates the notochord (nc); in panel E, the magenta arrow highlights the reduced notochord and disorganized floor plate.</p

Inducible transgene expression in adult zebrafish.

<p>(<b>A–D</b>) Expression of EGFP in the eye of a single adult <i>Tg(krt5:Gal4-ERT-VP16; UAS:EGFP)</i> zebrafish prior to (A and B) and after (C and D) treatment with 1 μM tamoxifen for one hour per day for three consecutive days. Green arrows point to EGFP expression in the eye. The areas bounded by the dashed red box in A and C are shown at high magnification in B and D, respectively. (<b>E–H</b>) Immunostaining of paraffin sections with anti-EGFP antibodies (shown in green) in eyes from DMSO- (E and F) and tamoxifen-treated (G and H) <i>Tg(krt5:Gal4-ERT-VP16; UAS:EGFP)</i> fish. Panels F and H show overlays with anti-EGFP antibody staining in green, Hoechst-stained nuclei in blue, and auto-fluorescence in red. Fish were drug treated as in A–D. Green arrows indicate EGFP expression in photoreceptors and asterisks (*) denote auto-fluorescence in photoreceptor outer segments. (<b>I</b>) In situ hybridization for <i>EGFP</i> mRNA in a paraffin section from a 4-OHT treated <i>Tg(krt5:Gal4-ERT-VP16; UAS:EGFP)</i> animal. The blue arrow shows <i>EGFP</i> expression in photoreceptors.</p

The Gal4-ERT system provides rapid induction of gene expression.

<p>(<b>A–D</b>) Kinetics of EGFP expression in <i>Tg(krt5:Gal4-ERT-VP16; UAS:EGFP)</i> animals upon administration of 2 μM 4-OHT at 24 hpf for 0–4.5 hours. (<b>A′–D′</b>) High magnification images demonstrating EGFP expression for each treatment. White arrowheads point to epidermal expression of EGFP; blue arrows indicate bleed-through from the heart muscle-specific ECFP transgenesis marker.</p

Temporally and spatially controlled transgene expression in zebrafish.

<p>(<b>A</b>) Schematic of transgenic constructs used in the Gal4-ERT system. A tamoxifen-responsive Gal4-ERT-VP16 construct is expressed from a tissue-specific promoter that activates any UAS-linked responder line (shown here as a <i>5xUAS:EGFP</i> reporter) upon tamoxifen or 4-OHT exposure (orange squares). The <i>myl7:ECFP</i> cassette serves as a transgenesis marker for the Gal4-ERT lines. (<b>B–D</b>) Visualization of EGFP expression in <i>Tg(krt5:Gal4-ERT-VP16; UAS:EGFP)</i> animals treated with ethanol (B) or 2 μM 4-OHT (C and D) from 4–24 hpf. The white arrow in panel D highlights expression of EGFP in the epidermis. (<b>E–I</b>) EGFP expression in <i>Tg(dusp6:Gal4-ERT-VP16; UAS:EGFP)</i> animals treated with vehicle (E) or 2 μM 4-OHT (F-I) from 4–24 hpf. In panels G and H, the white arrow indicates EGFP expression in the hindbrain and midbrain-hindbrain boundary. In panel I, arrowheads mark dorsal spinal cord neurons and the arrow points to EGFP expression in the floor plate. (<b>J–M</b>) Expression of EGFP in control (J) and 4-OHT treated (2 μM, K-M) <i>Tg(ef1α:Gal4-ERT-VP16; UAS:EGFP)</i> animals in a variety of cell types throughout the embryo including skeletal muscle (K, white arrow), the eye (L, white arrow), and the midbrain/midbrain-hindbrain boundary (M, white arrow). In panels B, E, and J, blue arrows point to myocardial ECFP expression, which represents the marker for transgenesis and serves as an internal control.</p

Using inducible expression to define temporal roles of Notch1 signaling in notochord development.

<p>(<b>A–E</b>) Expression of myc-tagged NICD by immunostaining with myc antibodies on 24 hpf <i>Tg(dusp6:Gal4-ERT-VP16; UAS:NICD)</i> embryos treated with ethanol (A) or 4 μM 4-OHT at the indicated times (B–E). Blue arrows indicate myocardial ECFP expression to mark transgenic animals and magenta arrows show expression of myc-tagged NICD. Panel insets display high magnification images of boxed regions where red arrows indicate the notochord (A and E) and the missing notochord (B–D). (<b>F</b>) Normalized penetrance of notochord defects in <i>Tg(dusp6:Gal4-ERT-VP16; UAS:NICD)</i> animals treated with 4-OHT at the indicated stages of development. Each data point represents the normalized fraction of affected animals in sets of treated embryos from four independent clutches. The data is normalized to the average fraction of abnormal fish in the four 4–24 h sets. The double asterisk indicates a significant difference between 4–24 h and 10–24 h 4-OHT treated fish (P<0.005). (<b>G–I</b>) Expression of <i>shha</i> in the floor plate of animals treated with ethanol (G) or with 4-OHT for the indicated times (H–I). Boxed regions are shown at higher magnification in the panel insets. Red arrows indicate <i>shha</i> expressing floor plate cells.</p

Transgene expression levels depend upon 4-OHT dosage.

<p>(<b>A–D</b>) EGFP expression upon treatment of <i>Tg(krt5:Gal4-ERT-VP16; UAS:EGFP)</i> zebrafish with ethanol or the indicated dose of 4-OHT from 4–24 hpf. The blue arrow indicates <i>myl7:ECFP</i> expression. (<b>A′–D′</b>) High-magnification images of ventral epidermis from fish in each treatment group. (<b>E</b>) Normalized EGFP intensity (to the 0.5 μM 4-OHT treated group) of fish treated with ethanol or 4-OHT. Error bars represent standard deviations.</p