Elevated levels of low density lipoprotein cholesterol (LDL-C) in plasma are a major contributor to cardiovascular disease (CVD), which is the leading cause of death worldwide. Genome–wide association studies (GWAS) have identified 95 loci that associate with control of lipid/cholesterol metabolism. Although GWAS results are highly provocative, direct analyses of the contribution of specific allelic variations in regulating LDL-C has been challenging due to the difficulty in accessing appropriate cells from affected patients. The primary cell type responsible for controlling cholesterol and lipid flux is the hepatocyte. Recently we have shown that cells with hepatocyte characteristics can be generated from human induced pluripotent stem cells (iPSC). This finding raises the possibility of using patient–specific iPSC–derived hepatocytes to study the functional contribution of GWAS loci in regulating lipid metabolism. To test the validity of this approach we produced iPSCs from a patient with mutations in the Low density lipoprotein receptor(LDLR) gene that result in familial hypercholesterolemia (FH). Conclusion: We demonstrate that 1) hepatocytes can be efficiently generated from FH iPSCs, 2) in contrast to control cells FH iPSC–derived hepatocytes are deficient in LDL–C uptake, 3) control but not FH iPS cell–derived hepatocytes increase LDL uptake in response to lovastatin, and 4) FH iPSC–derived hepatocytes display a marked elevation in secretion of lipidated ApoB-100. Cumulatively, these findings demonstrate that FH iPSC–derived hepatocytes recapitulate the complex pathophysiology of FH in culture. These results also establish that patient specific iPSC–derived hepatocytes could be used to definitively determine the functional contribution of allelic variation in regulating lipid and cholesterol metabolism and could potentially provide a platform for the identification of novel treatments of CVD.

The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, Bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals—but not all—develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

BMP signaling is fundamental to development, injury response, and homeostasis. We have developed transgenic zebrafish that report Smad-mediated BMP signaling in embryos and adults. These lines express either eGFP, destabilized eGFP, or destabilized KO2 under the wellcharacterized 'BMP Response Element' (BRE). These fluorescent proteins were found to be expressed dynamically in regions of known BMP signaling including the developing tailbud, hematopoietic lineage, dorsal eye, brain structures, heart, jaw, fins, and somites, as well as other tissues. Responsiveness to changes in BMP signaling was confirmed by observing fluorescence after activation in an hsp70:bmp2b transgenic background or by inhibition in an hsp70:nog3 background. We further demonstrated faithful reportage by the BRE transgenic lines following chemical repression of BMP signaling using an inhibitor of BMP receptor activity, dorsomorphin. Overall, these lines will serve as valuable tools to explore the mechanisms and regulation of BMP signal during embryogenesis, in tissue maintenance, and during disease.

HtrA-type serine proteases participate in folding and degradation of aberrant proteins and in processing and maturation of native proteins. Mutation of the corresponding genes often confers a pleiotropic phenotype that can include temperature sensitivity, sensitivity to osmotic and oxidative stress, and attenuated virulence. There are three HtrA-type serine proteases, YkdA, YvtA, and YycK, encoded in the Bacillus subtilis genome. In this report we show that YkdA and YvtA display many similarities: their expression profiles during the growth cycle in wild-type and mutant backgrounds are very alike, with expression being directed by very similar promoters. Both are induced by temperature upshift and by heterologous amylases at the transition phase of the growth cycle. These characteristics are quite different for YycK, suggesting that it has a cellular function distinct from that of the other two proteases or that it performs the same function but under different conditions. We also show that inactivation of either ykdA or yvtA results in compensating overexpression of the other gene, especially during stress conditions, with a concomitant increase in resistance to heat and hydrogen peroxide stresses. Mutation of both ykdA and yvtA leads to growth defects and to thermosensitivity. The fact that their expression increases dramatically at the transition phase of the growth cycle under certain conditions suggests that the YkdA and YvtA proteases may function in the processing, maturation, or secretion of extracellular enzymes in B. subtilis.Members of the HtrA family of serine proteases are widely distributed in nature, from bacteria to humans (10). The proteins are characterized by an amino-terminal domain that participates in protein localization, a catalytic domain containing an active serine residue, and a PDZ domain that functions in multimerization of the protein into the active dodecamer structure and perhaps also in identification of target proteins. Information derived from completely sequenced genomes shows that most eubacteria have a single HtrA-like serine protease. A recognizable member of the HtrA-protease family has been identified only in some archaebacteria, while the very small genomes of Mycoplasma pneumoniae and Mycoplasma genitalium do not appear to encode such a protease. However, a significant number of bacterial genomes encode more than one HtrA-like serine protease. Mycobacterium tuberculosis has four such genes; Escherichia coli, Bacillus subtilis, Treponema pallidum, Deinococcus radiodurans, and Synechocystis each have three copies, while Haemophilus influenzae and Pseudomonas aeruginosa each have two copies. These observations prompt questions about the physiological roles of each paralogue and of the extent to which their functions overlap.A comparison of the amino-terminal and PDZ domain regions of paralogues from each bacterium suggests a significant functional divergence. E. coli and T. pallidum each have two HtrA-like proteases with two PDZ domains and one HtrA-like protease with a si...

The retinal pigment epithelium (RPE) is a specialized monolayer of pigmented cells within the eye that is critical for maintaining visual system function. Diseases affecting the RPE have dire consequences for vision, and the most prevalent of these is atrophic (dry) age-related macular degeneration (AMD), which is thought to result from RPE dysfunction and degeneration. An intriguing possibility for treating RPE degenerative diseases like atrophic AMD is the stimulation of endogenous RPE regeneration; however, very little is known about the mechanisms driving successful RPE regeneration in vivo. Here, we developed a zebrafish transgenic model (rpe65a:nfsB-GFP) that enabled ablation of large swathes of mature RPE. RPE ablation resulted in rapid RPE degeneration, as well as degeneration of Bruch's membrane and underlying photoreceptors. Using this model, we demonstrate for the first time that larval and adult zebrafish are capable of regenerating a functional RPE monolayer after RPE ablation. Regenerated RPE cells first appear at the periphery of the RPE, and regeneration proceeds in a peripheral-to-central fashion. RPE ablation elicits a robust proliferative response in the remaining RPE. Subsequently, proliferative cells move into the injury site and differentiate into RPE. BrdU pulse-chase analyses demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological inhibition of Wnt signaling significantly reduces cell proliferation in the RPE and delays overall RPE recovery. These data demonstrate that the zebrafish RPE possesses a robust capacity for regeneration and highlight a potential mechanism through which endogenous RPE regenerate in vivo. SIGNIFICANCE STATEMENTDiseases resulting in RPE degeneration are among the leading causes of blindness worldwide, and no therapy exists that can replace RPE or restore lost vision. One intriguing possibility is the development of therapies focused on stimulating endogenous RPE regeneration. For this to be possible, we must first gain a deeper understanding of the mechanisms underlying RPE regeneration. Here, we ablate mature RPE in zebrafish and demonstrate that zebrafish regenerate RPE after widespread injury. Injury-adjacent RPE proliferate and regenerate RPE, suggesting that they are the source of regenerated tissue. Finally, we demonstrate that Wnt signaling is required for RPE regeneration. These findings establish an in vivo model through which the molecular and cellular underpinnings of RPE regeneration can be further characterized.