A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function.

The foregut endoderm gives rise to several organs including liver, pancreas, lung and thyroid with important roles in human physiology. Understanding which genes and signalling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to develop a rapid and scalable CRISPR/Cas-based mutagenesis strategy aiming at the identification of genes involved in morphogenesis and function of the thyroid. Core elements of the mutagenesis assay comprise bi-allelic gene invalidation in somatic mutants, a non-invasive monitoring of thyroid development in live transgenic fish, complementary analyses of thyroid function in fixed specimens and quantitative analyses of mutagenesis efficiency by Illumina sequencing of individual fish. We successfully validated our mutagenesis-phenotyping strategy in experiments targeting genes with known functions in early thyroid morphogenesis (pax2a, nkx2.4b) and thyroid functional differentiation (duox, duoxa, tshr). We also demonstrate that duox and duoxa crispants phenocopy thyroid phenotypes previously observed in human patients with bi-allelic DUOX2 and DUOXA2 mutations. The proposed combination of efficient mutagenesis protocols, rapid non-invasive phenotyping and sensitive genotyping holds great potential to systematically characterize the function of larger candidate gene panels during thyroid development and is applicable to other organs and tissues.

Developmental regulation of gene expression in the thyroid gland of Xenopus laevis tadpoles.

Thyroid hormones (TH) are the primary morphogen regulating amphibian metamorphosis. However, knowledge about molecular mechanisms regulating thyroid gland activity in anuran tadpoles is very scarce. In this study, we characterized gene expression profiles in thyroids of Xenopus laevis tadpoles during spontaneous metamorphosis. Using real-time PCR, elevated expression of slc5a5, tpo, tshr, and sar1a mRNAs was detected at late prometamorphic and climax stages. For dio2 and dio3 but not dio1, developmental regulation of thyroidal expression was evident from a strong up-regulation at late stages. Conversely, expression of the DNA replication markers mcm2 and pcna declined at climax stages. The presence of functional feedback mechanisms at premetamorphic stages was examined in two experiments. Stage 52 tadpoles were exposed for 72 h to 1.0 microg/l thyroxine (T4). This treatment caused reduced mRNA expression of slc5a5, tpo, and dio2, whereas no significant changes were detectable for tshr expression in thyroids and tshb expression in the pituitary. In another experiment, stage 46 tadpoles were treated with 20 mg/l sodium perchlorate (PER) for 5 and 10 days. Within this period of time, control tadpoles developed to stages 50 and 52, respectively. PER treatment resulted in up-regulation of slc5a5, tpo, and tshr mRNAs at both time points and increased dio2 mRNA expression at day 10. Effects of PER on thyroid histology were only apparent on day 10. Together, our analyses of thyroidal gene expression demonstrate a marked developmental regulation for functional markers of thyroid activity, two deiodinases as well as for DNA replication markers. Expression patterns detected in PER- and T4-treated tadpoles indicate that functional feedback signaling controlling thyroid activity is already active during premetamorphosis.

Perchlorate and ethylenethiourea induce different histological and molecular alterations in a non-mammalian vertebrate model of thyroid goitrogenesis.

Despite evidence for a conserved role of thyroid-stimulating hormone (TSH) in regulating vertebrate thyroid function, molecular data on thyroid responses to TSH are mainly limited to mammalian species. In this study, we examined histological and molecular changes in the thyroid of Xenopus laevis tadpoles during a 12-day treatment with 20mg/l perchlorate (PER) and 50mg/l ethylenethiourea (ETU). Inhibition of thyroid hormone (TH) synthesis by PER and ETU was evident from developmental retardation, reduced expression of TH-regulated genes and up-regulation of tshb-A mRNA. Thyroid histopathology revealed goiters with strikingly different follicular morphologies following PER and ETU treatment. Using real-time PCR, we analyzed thyroids sampled on day 12 for differential expression of 60 candidate genes. Further temporal analyses were performed for a subset of 14 genes. Relative to the control, PER and ETU treatment modulated the expression of 51 and 49 transcripts, respectively. Particularly genes related to TH synthesis and protein metabolism were similarly affected by PER and ETU. However, several genes were differentially expressed in PER- and ETU-treated tadpoles. Specifically, goiter formation in the PER treatment was associated with low expression of genes related to DNA replication but high expression of negative growth regulators. Results from this work provide for the first time a characterization of gene expression profiles during goitrogenesis in a non-mammalian vertebrate model. Overall, our data suggest that, in addition to TSH over-stimulation, further mechanisms related to the mode of goitrogen action contribute to the regulation of thyroid gene expression.

Luteinizing hormone, follicle stimulating hormone, and gonadotropin releasing hormone mRNA expression of Xenopus laevis in response to endocrine disrupting compounds affecting reproductive biology.

Environmental pollutants can interfere with the endocrine system of a variety of animals and are suggested to contribute to the worldwide decline of amphibians. In this study, the effects of endocrine disrupting compounds (EDC) on the hypothalamus-pituitary-gonad axis, regulating reproduction, were investigated in Xenopus laevis by determining their potential impact on gene expression of gonadotropin releasing hormone (GnRH), luteinizing hormone beta-subunit (LHbeta) and follicle-stimulating hormone beta-subunit (FSHbeta) in brain and pituitary using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). One environmental sample and four model compounds, ethinylestradiol (EE2), tamoxifen (TAM), methyldihydrotestosterone (MDHT), and flutamide (FLU), corresponding to (anti)estrogenic and (anti)androgenic modes of action were used at 10(-8)M during a four weeks exposure of adults of both sexes. In general, males had a higher LHbeta mRNA level compared to females, while the mRNA expression of FSHbeta and GnRH did not differ between both sexes. EE2 and MDHT treatment decreased LHbeta mRNA expression in the brain of male X. laevis, while only EE2 but not MDHT reduced LHbeta mRNA in females indicating classical negative feed-back mechanisms on hypophyseal gonadotropin expression. TAM increased LHbeta mRNA and FSHbeta mRNA expression in female X. laevis while none of the other treatments showed an effect on FSHbeta mRNA expression. GnRH expression was not changed by any treatment and exposure of X. laevis to Lambro river water had no significant effect on any of the genes examined. It is reported for the first time in amphibians that gonadotropin mRNA expression is differentially regulated by (anti)estrogenic and (anti)androgenic EDC and that gender-specific patterns of gene expression exist.

Structure of freely suspended chiral smectic films as determined by x-ray reflectivity and optical ellipsometry.

We report on a combined x-ray reflectivity and optical ellipsometry study of freely suspended smectic (Sm) films of a chiral liquid crystalline compound with the phase sequence Sm-A-Sm-C(*)(alpha)-Sm-C*-Sm-C(*)(gamma)-Sm-C(*)(A). Using tilt magnitude profiles from x-ray reflectivity as input to model the average optical properties obtained by ellipsometry, tilt direction profiles are also obtained. In this way realistic models can be elaborated for the various types of chiral Sm-C films. We find that the surface layers are more tilted than the interior layers due to surface interactions and finite size effects. For the ferrielectric Sm-C(*)(gamma) phase the tilt direction profile corresponds to a three-layer helix, in agreement with the clock model of chiral Sm-C phases. In thin films the surface interactions suppress the bulk helix structure of the Sm-C(*)(alpha) phase and a Sm-C(*)(A)-like structure is formed with an anticlinic layer-by-layer alternation of the tilt directions.

Crystalline smectic-B films as fluctuating systems: static and dynamic x-ray scattering.

Static and dynamic x-ray scattering has been used to characterize thermal fluctuations in free-standing smectic films in the crystalline-B phase. The results are remarkably similar to those in smectic-A films, in spite of the three-dimensional positional order in the crystalline-B phase. The main difference is the disappearance of the characteristic Landau-Peierls instability of the smectic-A phase. The dynamic nature of the fluctuations is explicitly demonstrated by x-ray photon correlation spectroscopy. This fluctuation behavior of crystalline-B films can be attributed to the small value of the shear elastic constant C44.

A procedure to secure a jugular vein catheter system onto the neck of the hamster.

We describe a procedure to secure a jugular vein catheter system at the dorsal nape of the neck in the hamster. An 8-cm piece of silicone tubing is connected with a 2.6 cm L-shaped metal tubing which is embedded in prosthetic material. The prosthetic material is placed underneath the neck skin of the hamster and keeps the metal end of the catheter system in a sturdy, upright position.

Inhibitors of ser/thr phosphatases 1 and 2A induce apoptosis in pituitary GH3 cells.

Two structurally different inhibitors of ser/thr phosphatases 1 and 2A, okadaic acid and calyculin A, time- and concentration-dependently stimulated and inhibited cell-specific function (hormone gene expression) in pituitary GH3 cells. The negative effect was associated with the appearance of apoptotic cell death. Nanomolar concentrations of both agents produced the characteristic morphological alterations and a DNA fragmentation ladder. Calyculin A treatment resulted in comparable changes with 10fold lower concentrations than okadaic acid. Observations with derivatives of okadaic acid with no or lower phosphatase inhibitory potency supported the conclusion that apoptosis induction is related to inhibition of ser/thr phosphatases, presumably types 1 and 2A. Membrane damage as measured by lactate dehydrogenase liberation into medium was significantly lower in apoptotic vs. necrotic cells. DNA fragmentation could be reduced by the addition of zinc but not by removal of extracellular calcium with EGTA. Apoptotic changes were reduced by the concomitant activation of protein kinase A by a membrane permeable cAMP analogue. Incubation of cells for 4 months in successively increased concentrations of okadaic acid resulted in a population that proliferated at the initially lethal concentration of 30 nM.

Effect of dietary magnesium level on urinary and faecal excretion of calcium, magnesium and phosphorus in adult, ovariectomized cats.

Dietary Mg restriction is generally considered to contribute to the prevention of struvite urolithiasis in cats, but its effects on faecal and urinary excretion of Ca and P have not been systematically investigated. The present study seeks to fill the gap. In a 4 x 4-week crossover study, ovariectomized cats were fed on purified diets containing either 0.40, 0.79, 1.59 or 3.17 mmol Mg/MJ (0.19, 0.38, 0.76 and 1.52 g Mg/kg diet). Increasing the dietary Mg level from 0.40 to 3.17 mmol Mg/MJ by the addition of extra MgCO3 raised urinary and faecal excretion of Mg from 0.14 to 0.68 mmol/MJ and from 0.28 to 1.66 mmol/MJ respectively. The 8-fold increase in Mg intake significantly raised urinary excretion of Ca from 0.06 to 0.09 mmol/MJ. Apparent absorption and urinary excretion of P were depressed by 13 and 25% respectively when the dietary Mg level was raised from 0.40 to 3.17 mmol/MJ. A dietary level of 0.40 mmol Mg/MJ (0.19 g Mg/kg diet) was found to be sufficient to maintain Mg balance in the adult ovariectomized cats fed on the purified diet.

Dietary phosphorus restriction to half the minimum required amount slightly reduces weight gain and length of tibia, but sustains femur mineralization and prevents nephrocalcinosis in female kittens.

The effects of dietary P restriction to half the recommended minimum level on growth, bone and renal mineralization and urinary composition were studied in female kittens. In two separate experiments, 8-week-old weanling kittens were fed on purified diets containing either 4.6 or 9.2 mmol P/MJ (2.8 or 5.6 g P/kg diet). In the second experiment there was an additional low-P diet in which the Ca concentration was reduced from 9.5 to 4.8 mmol/MJ (7.5 v. 3.8 g Ca/kg diet). P restriction slightly but systematically reduced weight gain (to a maximum of 16%) and growth of the tibia (by 1-4%); the former effect was statistically significant (P < 0.05) between the ages of 15 and 20 weeks in Expt 1 only, and the latter did not reach statistical significance at any time point (P > or = 0.13). No adverse effect of P restriction was found on mineralization of femur at the age of 39 weeks. Kidney Ca concentrations were significantly lowered (Expt 1, 6 v. 20 mumol/g dry weight, P < 0.001; Expt 2, 7 v. 16 mumol/g dry weight, P < 0.01) in cats fed on the low-P diets, this effect not being affected by the dietary Ca:P ratio. Urinary P concentration was significantly depressed (by 50-96%) after feeding the low-P diets (P < 0.001). P intake did not influence P, Ca and Mg retention during the period of 15 to 39 weeks of age.

Dietary calcium chloride vs. calcium carbonate reduces urinary pH and phosphorus concentration, improves bone mineralization and depresses kidney calcium level in cats.

The effect of dietary calcium chloride vs. calcium carbonate on mineral metabolism was studied in cats. Ovariectomized cats and female kittens were fed purified diets with a normal calcium level (9.5 mmol Ca/MJ) but containing either calcium carbonate or calcium chloride, or were fed diets with a high calcium level (17.7 mmol Ca/MJ) containing either calcium carbonate alone or equimolar amounts of both calcium carbonate and calcium chloride. A 4 x 4-wk cross-over study using adult cats and a 31-wk parallel study using kittens were conducted. Calcium, phosphorus and magnesium balances were established regularly. In the course of the experiment with the kittens, blood samples were taken and X-ray photographs of the tibiae made. At the age of 39 wk, the kittens were killed, and organs and bones were collected. In both adult cats and kittens fed the high calcium diets, urinary concentrations of magnesium and phosphorus and apparent absorption of these minerals were lower than after feeding the normal calcium diets. Urinary pH and phosphorus concentration were lower in cats and kittens fed diets with calcium chloride instead of calcium carbonate. Body weight gain and tibia growth in the kittens tended to be greater after feeding the diets with calcium chloride. Calcium chloride vs. calcium carbonate and also supplemental calcium chloride in the high calcium diet significantly stimulated femur density and reduced renal calcium concentration.

Substitution of dietary calcium chloride for calcium carbonate reduces urinary ph and urinary phosphorus excretion in adult cats.

In a 4x4-wk cross-over study, eight adult cats were given four moist diets containing identical amounts of calcium (13.9 mmol/MJ) but with different ratios of calcium carbonate to calcium chloride, the calcium salts providing half of the total dietary calcium. Increasing amounts of calcium chloride were substituted for equimolar amounts of calcium carbonate. Higher intakes of calcium chloride caused significantly lower pH values in postprandial and 24-h urine samples. The urinary excretion of ammonium and titratable acid rose with increasing calcium chloride intake. The urinary concentrations of calcium and magnesium were not affected by the type of calcium salt, but the urinary excretion and concentration of phosphorus were significantly depressed when the amount of calcium chloride in the diet was increased. The results are discussed in the context of dietary prevention of and therapy for struvite urolithiasis in cats.

Estimation of shear stress-related blood damage in heart valve prostheses--in vitro comparison of 25 aortic valves.

The hemodynamics of heart valve prostheses can be reproducibly investigated in vitro within circulatory mock loops. By measuring the downstream velocity and shear stress fields the shear stresses which are clinically responsible for damage to platelets and red blood cells can be determined. The mechanisms of damage and the effects of shear stresses on blood corpuscles were investigated by Wurzinger et al. at the Aerodynamics Institute of the RWTH Aachen. In the present study, the above data are incorporated into a mathematical correlation, which serves as a basic model for the estimation of blood damage. This mathematical model was applied to in vitro investigations of 25 different aortic valve prostheses. The results were compared to clinical findings. In most cases agreement was good, indicating that this model may be directly applied to the clinical situation. This new method facilitates the estimation of clinically expected blood damage from in vitro measurements. It may be useful for the development and evaluation of new valve prostheses. By comparative evaluation of different valve types it also provides additional information to help the implanting surgeon select the optimum valve for his patient.

Ultrastructural investigations on the question of mechanical activation of blood platelets.

The present study addresses the question whether platelets are activated by mechanical stresses as they occur in pathologically accelerated blood flow. Their potential mechanoreceptive properties were tested by subjecting human platelets to defined fluid mechanical forces for periods of milliseconds. Platelet activation was assessed by quantitative morphology, revealing besides activated platelets, irreversibly ballooned, lytic platelets. However, this morphometrically documented "shear activation" of platelets can be suppressed almost completely by the addition of enzyme-substrate systems, capable of removing adenosine diphosphate from the suspending medium. This is in keeping with a recent study from our laboratory showing that the behaviour of lactic dehydrogenase as marker for platelet lysis and beta-thromboglobulin as release marker refuted earlier data, suggesting a direct activation of platelets by shear. It is concluded that former evidence of "shear induced platelet activation" must be interpreted as the consequence of lytic damage to a small proportion of platelets.

Platelet and coagulation parameters following millisecond exposure to laminar shear stress.

Blood flowing through artificial organs or arterial stenoses is subjected to high shear for short times. To clarify the effects of short acting high shear forces upon platelets, heparinized PRP was exposed to viscometric flow (57-255 N/m2; 7-700 ms). Before and after shear exposure beta-TG release, LDH liberation, platelet count, and ADP-induced aggregation were assayed. Stypven time-monitored platelet procoagulant activity, determined after heparin neutralization by protamine, proved to be the most sensitive indicator for shear-induced platelet alteration. A shear stress of 170 N/m2, acting for as short a time as 7 ms made available procoagulant phospholipids, whereas beta-TG and LDH liberation required 255 N/m2 for 7 ms, and ADP refractoriness was found only after 113 ms exposure to 255 N/m2. Under these conditions the percent release of beta-TG does not exceed the liberation of LDH, suggesting that the evidence for "shear induced platelet activation" from experiments with exposure times of minutes, is most likely a conventional biochemical rather than biophysical effect.

"Shear induced platelet activation"--a critical reappraisal.

Platelets were found to be stimulated by high shear exposure for 5 minutes. Using a new technique, shear stresses up to 255 N/m2, acting for pathophysiological relevant intervals of milliseconds did not elicit active release of beta-TG, beyond the amount explained by LDH-monitored passive lysis. Neither the plasma level of ionized calcium (citrate vs heparin anticoagulation), nor a potent platelet inhibiting agent like PgI2 (20 nM) did significantly alter platelet responses to short acting high shear stress. Ultrastructural signs of activation could largely be suppressed by adding ADP-scavengers. Direct "shear induced platelet activation" appears rather unlikely and mostly reducible to secondary biochemical activation by mediators, probably adenine nucleotides, from a small percentage of passively shear-destroyed platelets. The extent of this secondary activation is largely a matter of experimental conditions.