Transgenic Wnt/TCF pathway reporters: all you need is Lef?

The Wnt signaling pathway controls a large and diverse set of cell fate decisions in embryonic development, adult organ maintenance and disease. At the transcriptional level, Wnt/beta-catenin signaling is primarily mediated by the T-cell factor (TCF)/Lef-1 family of transcription factors, referred to here as TCFs. In order to track Wnt pathway activity during animal development, several laboratories have built transgenic reporter constructs containing multimerized TCF binding sites. Most of these reporters are active at multiple known sites of Wnt signaling, and several act as faithful reporters of pathway activity in specific contexts. However, multimerized TCF reporters should not be assumed to give a complete or definitive readout of Wnt signaling in vivo. Direct comparisons reveal discrepancies among reporters; in addition, there is good reason to expect that some important types of pathway activity, including target gene de-repression and TCF-independent Wnt or beta-catenin signaling, will not be accurately reported by such constructs. This review will discuss various transgenic Wnt/beta-catenin/TCF reporters, address the fidelity and completeness of their Wnt responsiveness, and contrast their in vivo transcriptional responses with those of natural Wnt target genes. Finally, three caveats to the interpretation of multimerized TCF reporter expression patterns will be proposed.

Angiotensin I-converting enzyme genotype significantly affects progression of IgA glomerulonephritis in an italian population.

To evaluate the role of angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism in the progression of immunoglobulin A glomerulonephritis (IgA-GN), genotype distribution in 81 biopsy-proven cases of IgA-GN was studied. A logistic regression model showed that the risk for homozygous DD was not significantly elevated in patients with IgA-GN compared with healthy subjects (odds ratio = 1.16; confidence interval [CI], 0.4 to 3.3). However, the 5-year (78% v 90%) and 10-year (52% v 82%) renal survival rates for 47 patients with serum creatine (Cr) levels of 1.5 mg/dL or less at biopsy was significantly less in DD patients (n = 18; chi2 = 5.41; P = 0.02). The hazard ratio (HR) for DD (multivariate analysis from Cox proportional model after adjustment for known factors of progression, such as hypertension [HPT] and proteinuria [PTO]) was 3.07 (CI, 1.1 to 9.4). The HR for heavy PTO was 6.1 (CI, 1.9 to 19). The association of DD genotype with progression was even more striking when patients with other risk factors (heavy proteinuria) were excluded, as shown by DD-related risk in the absence (HR = 3.6; CI, 1.1 to 12) and presence (HR = 2; CI, 0.4 to 10) of PTO. The risk ratio was further increased by the coexistence of DD + PTO (HR = 9.16; CI, 1.8 to 15.7). Furthermore, in a cross-sectional study among patients with IgA-GN, a logistic regression model showed that the risk for homozygous DD was greater, although not at a statistically significant level in the end-stage renal failure subgroup compared with the normal renal function subgroup (odds ratio = 3.16; CI, 0.7 to 13.7) after adjustment by sex, age at biopsy, HPT, PTO, and therapy. Last, DD was significantly more frequent in those patients who started hemodialysis at an earlier age (chi2 for trend = 6.81; P = 0.009). Our study further supports that ACE genotype is a risk factor not for the development, but for the worsening of IgA-GN clinical course. However, on the basis of current knowledge, we cannot exclude that I/D polymorphism may simply serve as a prognostic marker, eventually linked with other discrete loci involved in the progression of renal damage.

Correlations between bone histopathology and serum biochemistry in uremic patients on chronic hemodialysis.

To define which noninvasive investigations are of value in predicting bone histology, we analyzed transiliac bone specimens (66 biopsies, 14 autopsies) from 80 uremic patients on chronic dialysis. Results were compared with values of different measurements of parathyroid hormone (PTH), alkaline phosphatase (APH), osteocalcin, calcitonin, baseline and post-deferroxamine (DFO) aluminium (Al),--beta 2 microglobulin, ferritin and bone mineral density. Among histomorphometric parameters, woven osteoid, active osteoblastic surface and resorption surface showed the best correlations with dynamic and biochemical marks of active bone metabolism. Among biochemical parameters, intact PTH and APH were better related to histomorphometric and dynamic bone parameters than other PTH measurements as well as osteocalcin, while calcitonin was related to no parameters. Stainable Al alone, and not total bone Al content was related to bone histology. Baseline Al was related to lamellar osteoid, while post-DFO Al was related to stainable Al. beta 2 microglobulin was positively related to active osteoid surface and ferritin was inversely related to the mineral apposition rate, while bone mineral density was related only to total bone volume. We conclude that, though definite diagnosis requires the use of histological methods, few simple biochemical parameters may offer insight to the bone metabolic status, useful to the physician in day to day clinical practice.

hairy mediates dominant repression in the Drosophila embryo.

hairy encodes a bHLH repressor that regulates several developmental processes in Drosophila, including embryonic segmentation and neurogenesis. Segmentation repressors such as Krüppel and knirps have been shown to function over short distances, less than 50-100 bp, to inhibit or quench closely linked upstream activators. This mode of repression permits multiple enhancers to work independently of one another within a modular promoter. Here, we employ a transgenic embryo assay to present evidence that hairy acts as a dominant repressor, which can function over long distances to block multiple enhancers. hairy is shown to repress a heterologous enhancer, the rhomboid NEE, when bound 1 kb from the nearest upstream activator. Moreover, the binding of hairy to a modified NEE leads to the repression of both the NEE and a distantly linked mesoderm-specific enhancer within a synthetic modular promoter. Additional evidence that hairy is distinct from previously characterized embryonic repressors stems from the analysis of the gypsy insulator DNA. This insulator selectively blocks the hairy repressor, but not the linked activators, within a modified NEE. We compare hairy with previously characterized repressors and discuss the consequences of short-range and long-range repression in development.

Transcriptional repression in the Drosophila embryo.

Transcriptional repression is essential for the conversion of crude maternal gradients into sharp territories of tissue differentiation in the Drosophila embryo. Evidence will be presented suggesting that some of the embryonic repressors function through a short-range 'quenching' mechanism, whereby a repressor works over short distances (ca. 50 b.p.) to block neighbouring activators within a target enhancer. This type of repression can explain how different enhancers work autonomously within complex modular promoters. However, at least one of the repressors operating in the early embryo works through a long-range, or silencing, mechanism. The binding of a silencer to a given enhancer leads to the inactivation of all enhancers within a complex promoter. The analysis of chromatin boundary elements suggest that silencers and enhancers might work through distinct mechanisms. We speculate that silencers constrain the evolution of complex promoters.

The Fab-7 element of the bithorax complex attenuates enhancer-promoter interactions in the Drosophila embryo.

Enhancers integrate positive and negative regulatory information to direct localized patterns of gene expression in the Drosophila embryo. Here we present evidence for the occurrence of cis regulatory elements that control the levels of gene expression by modulating enhancer-promoter interactions. For this purpose we have investigated the Drosophila bithorax complex (BX-C) because genetic studies suggest that the BX-C contains as much as 300 kb of cis regulatory information. A specialized DNA element, Fab-7, has been proposed to function as a boundary element that separates the iab-6 and iab-7 cis regulatory regions within the Abd-B domain of the BX-C. A 1.2-kb Fab-7 DNA fragment was placed between divergently transcribed white and lacZ test promoters and challenged with several defined enhancers expressed in the early embryo. These studies suggest that Fab-7 functions as an attenuator, which weakens gene expression by reducing enhancer-promoter interactions. Fab-7 selectively blocks distal enhancers in an orientation-independent fashion, and can function when located far from either the distal enhancer or target promoter. Fab-7 may be related to insulator DNAs, which flank genetic loci and functionally isolate neighboring genes. We propose that specialized DNA elements, such as the Fab-7 attenuator, might play a general role in controlling the levels of gene expression by modulating enhancer-promoter interactions.

The eve stripe 2 enhancer employs multiple modes of transcriptional synergy.

Previous studies have provided a detailed model for the regulation of even-skipped (eve) stripe 2 expression in the Drosophila embryo. The bicoid (bcd) regulatory gradient triggers the expression of hunchback (hb); these work synergistically to activate the stripe in the anterior half of the embryo, bcd also coordinates the expression of two repressors, giant (gt) and Kruppel (Kr), which define the anterior and posterior borders of the stripe, respectively. Here, we report the findings of extensive cis- and trans- complementation analyses using a series of defective stripe 2 enhancers in transgenic embryos. This study reaches two primary conclusions. First, the strip 2 enhancer is inherently 'sensitized' for repression by gt. We propose that gt specifies the sharp anterior stripe border by blocking two tiers of transcriptional synergy, cooperative binding to DNA and cooperative contact of bound activators with the transcription complex. Second, we find that the synergistic activity of hb and bcd is 'promiscuous'. For example, a maternally expressed Gal4-Sp1 fusion protein can functionally replace hb in the stripe 2 enhancer. This finding challenges previous proposals for dedicated hb and bcd interactions in the segmentation process.

dCtBP mediates transcriptional repression by Knirps, Krüppel and Snail in the Drosophila embryo.

The pre-cellular Drosophila embryo contains 10 well characterized sequence-specific transcriptional repressors, which represent a broad spectrum of DNA-binding proteins. Previous studies have shown that two of the repressors, Hairy and Dorsal, recruit a common co-repressor protein, Groucho. Here we present evidence that three different repressors, Knirps, Krüppel and Snail, recruit a different co-repressor, dCtBP. Mutant embryos containing diminished levels of maternal dCtBP products exhibit both segmentation and dorsoventral patterning defects, which can be attributed to loss of Krüppel, Knirps and Snail activity. In contrast, the Dorsal and Hairy repressors retain at least some activity in dCtBP mutant embryos. dCtBP interacts with Krüppel, Knirps and Snail through a related sequence motif, PXDLSXK/H. This motif is essential for the repression activity of these proteins in transgenic embryos. We propose that dCtBP represents a major form of transcriptional repression in development, and that the Groucho and dCtBP co-repressors mediate separate pathways of repression.

The gap protein knirps mediates both quenching and direct repression in the Drosophila embryo.

Transcriptional repression is essential for establishing localized patterns of gene expression during Drosophila embryogenesis. Several mechanisms of repression have been proposed, including competition, quenching and direct repression of the transcription complex. Previous studies suggest that the knirps orphan receptor (kni) may repress transcription via competition, and exclude the binding of the bicoid (bcd) activator to an overlapping site in a target promoter. Here we present evidence that kni can quench, or locally inhibit, upstream activators within a heterologous enhancer in transgenic embryos. The range of kni repression is approximately 50-100 bp, so that neighboring enhancers in a modular promoter are free to interact with the transcription complex (enhancer autonomy). However, kni can also repress the transcription complex when bound in promoter-proximal regions. In this position, kni functions as a dominant repressor and blocks multiple enhancers in a modular promoter. Our studies suggest that short-range repression represents a flexible form of gene regulation, exhibiting enhancer- or promoter-specific effects depending on the location of repressor binding sites.

A notch-independent activity of suppressor of hairless is required for normal mechanoreceptor physiology.

Suppressor of Hairless [Su(H)]/Lag-1/RBP-Jkappa/CBF1 is the only known transducing transcription factor for Notch receptor signaling. Here, we show that Su(H) has three distinct functions in the development of external mechanosensory organs in Drosophila: Notch-dependent transcriptional activation and a novel auto-repression function, both of which direct cell fate decisions, and a novel auto-activation function required for normal socket cell differentiation. This third phase of activity, the first known Notch-independent activation function for Su(H) in development, depends on a cell type-specific autoregulatory enhancer that is active throughout adult life and is required for proper mechanoreception. These results establish a direct link between a broadly deployed cell signaling pathway and an essential physiological function of the nervous system.

The concept of 'glomerulonephritis'. the fascinating history of evolution and emergence of a specialist's nosology focus on Italy and Torino.

Though the term 'nephritis' first appeared in the 19th century, this word did not bear the same meaning as it does today; indeed, for many years it was used to indicate 'renal diseases' (in the sense of Bright's disease) in a larger sense. This review summarizes the long gestation of the concept of 'glomerulonephritis' from the prehistory of medicine up to the beginning of the second half of the 20th century with emphasis on Italy and, in particular, on Torino, which was the capital of the Kingdom of Italy from 1861 to 1865. To the best of our kowledge, this is the first study reporting an epidemiology survey of Bright's disease in Italy from 1880 up to 1960. Towards the end of the 19th century, Bright's disease accounted for 26 deaths/year/10(5) population (in comparison with more than 200 from tuberculosis) in Italy, roughly paralleling that reported in the USA. At the beginning of the 20th century, Bright's disease was the seventh cause of death (almost 1% of total deaths) in Italy. Furthermore, in Italy, as elsewhere, autopsy studies showed a higher percentage of deaths attributed to Bright's disease (5-7%) in comparison with those obtained from vital statistics. In 1960, just before the beginning of renal replacement therapy, Bright's disease accounted for 15.7 deaths/year/10(5) population (= 1.46% of all deaths), roughly paralleling that reported in the United Kingdom (13.8/10(5) population = 1.25% of deaths). Probably, it was difficult to recognize the real incidence of chronic renal diseases leading to death in the 1960s, and vital statistics were able to furnish only approximate estimates. However, noteworthy is the fact that these values were very close to those estimated as being the annual need for renal replacement therapy (10-20 cases/year/10(5) population).