Dynamic Interpretation of Hedgehog Signaling in the Drosophila Wing Disc

Morphogens are classically defined as molecules that control patterning by acting at a distance to regulate gene expression in a concentration-dependent manner. In the Drosophila wing imaginal disc, secreted Hedgehog (Hh) forms an extracellular gradient that organizes patterning along the anterior–posterior axis and specifies at least three different domains of gene expression. Although the prevailing view is that Hh functions in the Drosophila wing disc as a classical morphogen, a direct correspondence between the borders of these patterns and Hh concentration thresholds has not been demonstrated. Here, we provide evidence that the interpretation of Hh signaling depends on the history of exposure to Hh and propose that a single concentration threshold is sufficient to support multiple outputs. Using mathematical modeling, we predict that at steady state, only two domains can be defined in response to Hh, suggesting that the boundaries of two or more gene expression patterns cannot be specified by a static Hh gradient. Computer simulations suggest that a spatial “overshoot” of the Hh gradient occurs, i.e., a transient state in which the Hh profile is expanded compared to the Hh steady-state gradient. Through a temporal examination of Hh target gene expression, we observe that the patterns initially expand anteriorly and then refine, providing in vivo evidence for the overshoot. The Hh gene network architecture suggests this overshoot results from the Hh-dependent up-regulation of the receptor, Patched (Ptc). In fact, when the network structure was altered such that the ptc gene is no longer up-regulated in response to Hh-signaling activation, we found that the patterns of gene expression, which have distinct borders in wild-type discs, now overlap. Our results support a model in which Hh gradient dynamics, resulting from Ptc up-regulation, play an instructional role in the establishment of patterns of gene expression

Lateral Gene Expression in Drosophila Early Embryos Is Supported by Grainyhead-Mediated Activation and Tiers of Dorsally-Localized Repression

The general consensus in the field is that limiting amounts of the transcription factor Dorsal establish dorsal boundaries of genes expressed along the dorsal-ventral (DV) axis of early Drosophila embryos, while repressors establish ventral boundaries. Yet recent studies have provided evidence that repressors act to specify the dorsal boundary of intermediate neuroblasts defective (ind), a gene expressed in a stripe along the DV axis in lateral regions of the embryo. Here we show that a short 12 base pair sequence (“the A-box”) present twice within the ind CRM is both necessary and sufficient to support transcriptional repression in dorsal regions of embryos. To identify binding factors, we conducted affinity chromatography using the A-box element and found a number of DNA-binding proteins and chromatin-associated factors using mass spectroscopy. Only Grainyhead (Grh), a CP2 transcription factor with a unique DNA-binding domain, was found to bind the A-box sequence. Our results suggest that Grh acts as an activator to support expression of ind, which was surprising as we identified this factor using an element that mediates dorsally-localized repression. Grh and Dorsal both contribute to ind transcriptional activation. However, another recent study found that the repressor Capicua (Cic) also binds to the A-box sequence. While Cic was not identified through our A-box affinity chromatography, utilization of the same site, the A-box, by both factors Grh (activator) and Cic (repressor) may also support a “switch-like” response that helps to sharpen the ind dorsal boundary. Furthermore, our results also demonstrate that TGF-β signaling acts to refine ind CRM expression in an A-box independent manner in dorsal-most regions, suggesting that tiers of repression act in dorsal regions of the embryo

HLA-A and -B alleles and haplotypes in hemochromatosis probands with HFE C282Y homozygosity in central Alabama

BACKGROUND: We wanted to quantify HLA-A and -B allele and haplotype frequencies in Alabama hemochromatosis probands with HFE C282Y homozygosity and controls, and to compare results to those in other populations. METHODS: Alleles were detected using DNA-based typing (probands) and microlymphocytotoxicity (controls). RESULTS: Alleles were determined in 139 probands (1,321 controls) and haplotypes in 118 probands (605 controls). In probands, A*03 positivity was 0.7482 (0.2739 controls; p =< 0.0001; odds ratio (OR) 7.9); positivity for B*07, B*14, and B*56 was also increased. In probands, haplotypes A*03-B*07 and A*03-B*14 were more frequent (p < 0.0001, respectively; OR = 12.3 and 11.1, respectively). The haplotypes A*01-B*60, A*02-B*39, A*02-B*62, A*03-B*13, A*03-B*15, A*03-B*27, A*03-B*35, A*03-B*44, A*03-B*47, and A*03-B*57 were also significantly more frequent in probands. 37.3% of probands were HLA-haploidentical with other proband(s). CONCLUSIONS: A*03 and A*03-B*07 frequencies are increased in Alabama probands, as in other hemochromatosis cohorts. Increased absolute frequencies of A*03-B*35 have been reported only in the present Alabama probands and in hemochromatosis patients in Italy. Increased absolute frequencies of A*01-B*60, A*02-B*39, A*02-B*62, A*03-B*13, A*03-B*15, A*03-B*27, A*03-B*44, A*03-B*47, and A*03-B*57 in hemochromatosis cohorts have not been reported previously

Ancestral Vascular Lumen Formation via Basal Cell Surfaces

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals

Dpp Signaling Activity Requires Pentagone to Scale with Tissue Size in the Growing Drosophila Wing Imaginal Disc

The activity of the Dpp morphogen adapts to tissue size in the growing Drosophila wing imaginal disc, and Pentagone, an important secreted feedback regulator of the Dpp pathway, is required for this adaptation

Multifactorial Regulation of a Hox Target Gene

Hox proteins play fundamental roles in controlling morphogenetic diversity along the anterior–posterior body axis of animals by regulating distinct sets of target genes. Within their rather broad expression domains, individual Hox proteins control cell diversification and pattern formation and consequently target gene expression in a highly localized manner, sometimes even only in a single cell. To achieve this high-regulatory specificity, it has been postulated that Hox proteins co-operate with other transcription factors to activate or repress their target genes in a highly context-specific manner in vivo. However, only a few of these factors have been identified. Here, we analyze the regulation of the cell death gene reaper (rpr) by the Hox protein Deformed (Dfd) and suggest that local activation of rpr expression in the anterior part of the maxillary segment is achieved through a combinatorial interaction of Dfd with at least eight functionally diverse transcriptional regulators on a minimal enhancer. It follows that context-dependent combinations of Hox proteins and other transcription factors on small, modular Hox response elements (HREs) could be responsible for the proper spatio-temporal expression of Hox targets. Thus, a large number of transcription factors are likely to be directly involved in Hox target gene regulation in vivo

Transforming Growth Factor: β Signaling Is Essential for Limb Regeneration in Axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-β). In the present study, the full length sequence of the axolotl TGF-β1 cDNA was isolated. The spatio-temporal expression pattern of TGF-β1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-β signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-β type I receptor, SB-431542, we show that TGF-β signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-β signaling are down-regulated. These data directly implicate TGF-β signaling in the initiation and control of the regeneration process in axolotls

Effect of sitagliptin on cardiovascular outcomes in type 2 diabetes

BACKGROUND: Data are lacking on the long-term effect on cardiovascular events of adding sitagliptin, a dipeptidyl peptidase 4 inhibitor, to usual care in patients with type 2 diabetes and cardiovascular disease. METHODS: In this randomized, double-blind study, we assigned 14,671 patients to add either sitagliptin or placebo to their existing therapy. Open-label use of antihyperglycemic therapy was encouraged as required, aimed at reaching individually appropriate glycemic targets in all patients. To determine whether sitagliptin was noninferior to placebo, we used a relative risk of 1.3 as the marginal upper boundary. The primary cardiovascular outcome was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for unstable angina. RESULTS: During a median follow-up of 3.0 years, there was a small difference in glycated hemoglobin levels (least-squares mean difference for sitagliptin vs. placebo, -0.29 percentage points; 95% confidence interval [CI], -0.32 to -0.27). Overall, the primary outcome occurred in 839 patients in the sitagliptin group (11.4%; 4.06 per 100 person-years) and 851 patients in the placebo group (11.6%; 4.17 per 100 person-years). Sitagliptin was noninferior to placebo for the primary composite cardiovascular outcome (hazard ratio, 0.98; 95% CI, 0.88 to 1.09; P<0.001). Rates of hospitalization for heart failure did not differ between the two groups (hazard ratio, 1.00; 95% CI, 0.83 to 1.20; P = 0.98). There were no significant between-group differences in rates of acute pancreatitis (P = 0.07) or pancreatic cancer (P = 0.32). CONCLUSIONS: Among patients with type 2 diabetes and established cardiovascular disease, adding sitagliptin to usual care did not appear to increase the risk of major adverse cardiovascular events, hospitalization for heart failure, or other adverse events

The hemochromatosis 845 G-->A and 187 C-->G mutations: prevalence in non-Caucasian populations.

Hemochromatosis, the inherited disorder of iron metabolism, leads, if untreated, to progressive iron overload and premature death. The hemochromatosis gene, HFE, recently has been identified, and characterization of this gene has shown that it contains two mutations that result in amino acid substitutions-cDNA nucleotides 845 G-->A (C282Y) and 187 C-->G (H63D). Although hemochromatosis is common in Caucasians, affecting >=1/300 individuals of northern European origin, it has not been recognized in other populations. The present study used PCR and restriction-enzyme digestion to analyze the frequency of the 845 G-->A and 187 C-->G mutations in HLA-typed samples from non-Caucasian populations, comprising Australian Aboriginal, Chinese, and Pacific Islanders. Results showed that the 845 G-->A mutation was present in these populations (allele frequency 0.32%), and, furthermore, it was always seen in conjunction with HLA haplotypes common in Caucasians, suggesting that 845 G-->A may have been introduced into these populations by Caucasian admixture. 187 C-->G was present at an allele frequency of 2.68% in the two populations analyzed (Australian Aboriginal and Chinese). In the Australian Aboriginal samples, 187 C-->G was found to be associated with HLA haplotypes common in Caucasians, suggesting that it was introduced by recent admixture. In the Chinese samples analyzed, 187 C-->G was present in association with a wide variety of HLA haplotypes, showing this mutation to be widespread and likely to predate the more genetically restricted 845 G-->A mutation