Oncogenic cooperation between TCF7-SPI1 and NRAS(G12D) requires ß-catenin activity to drive T-cell acute lymphoblastic leukemia.

Spi-1 Proto-Oncogene (SPI1) fusion genes are recurrently found in T-cell acute lymphoblastic leukemia (T-ALL) cases but are insufficient to drive leukemogenesis. Here we show that SPI1 fusions in combination with activating NRAS mutations drive an immature T-ALL in vivo using a conditional bone marrow transplant mouse model. Addition of the oncogenic fusion to the NRAS mutation also results in a higher leukemic stem cell frequency. Mechanistically, genetic deletion of the ß-catenin binding domain within Transcription factor 7 (TCF7)-SPI1 or use of a TCF/ß-catenin interaction antagonist abolishes the oncogenic activity of the fusion. Targeting the TCF7-SPI1 fusion in vivo with a doxycycline-inducible knockdown results in increased differentiation. Moreover, both pharmacological and genetic inhibition lead to down-regulation of SPI1 targets. Together, our results reveal an example where TCF7-SPI1 leukemia is vulnerable to pharmacological targeting of the TCF/ß-catenin interaction.

Limb derived cells as a paradigm for engineering self-assembling skeletal tissues.

Mimicking developmental events has been proposed as a strategy to engineer tissue constructs for regenerative medicine. However, this approach has not yet been investigated for skeletal tissues. Here, it is demonstrated that ectopic implantation of day-14.5 mouse embryonic long bone anlagen, dissociated into single cells and randomly incorporated in a bioengineered construct, gives rise to epiphyseal growth plate-like structures, bone and marrow, which share many morphological and molecular similarities to epiphyseal units that form after transplanting intact long bone anlage, demonstrating substantial robustness and autonomy of complex tissue self-assembly and the overall organogenesis process. In vitro studies confirm the self-aggregation and patterning capacity of anlage cells and demonstrate that the model can be used to evaluate the effects of large and small molecules on biological behaviour. These results reveal the preservation of self-organizing and self-patterning capacity of anlage cells even when disconnected from their developmental niche and subjected to system perturbations such as cellular dissociation. These inherent features make long bone anlage cells attractive as a model system for tissue engineering technologies aimed at creating constructs that have the potential to self-assemble and self-pattern complex architectural structures.

Marine (Taeniura lymma) and freshwater (Himantura signifer) elasmobranchs synthesize urea for osmotic water retention.

The objective of this study was to elucidate whether the marine blue-spotted fantail ray, Taeniura lymma, and the freshwater white-edge whip ray, Himantura signifer, injected with NH(4)Cl intraperitoneally would excrete the majority of the excess ammonia as ammonia per se to ameliorate ammonia toxicity despite being ureogenic. To examine the roles of urea and the ornithine-urea cycle, experimental fishes were exposed to salinity changes after being injected with NH(4)Cl. The ammonia excretion rates of the marine ray, T. lymma, injected with NH(4)Cl followed by exposure to seawater (30 per thousand) or diluted seawater (25 per thousand) increased 13-fold and 10-fold, respectively, within the first 3 h. Consequently, the respective percentage of nitrogenous wastes excreted as ammonia were 55% and 65% compared with 21% of the saline-injected control, indicating that T. lymma became apparently ammonotelic after injection with NH(4)Cl. By hour 6, large portions (70%-85%) of the ammonia injected into T. lymma exposed to seawater or diluted seawater had been excreted, and T. lymma excreted much more nitrogenous wastes (135%-180%), in excess of the ammonia injected into the fish, during the 24-h period. For T. lymma exposed to seawater, a small portion (30%) of the ammonia injected into the fish was detoxified to urea during the first 6 h, but there was an apparent suppression of urea synthesis thereafter, contributing partially to the large decrease (19%) in urea contents in its muscle at hour 24. A major contributing factor to the decrease in urea content was a reduction in ammonia production, as indicated by a large deficit between urea loss in the muscle and excess ammonia accumulated plus excess nitrogen excreted in the experimental fish. The freshwater ray, H. signifer, injected with NH(4)Cl followed by exposure to freshwater (0.7 per thousand) or brackish water (10 per thousand) was capable of excreting all the ammonia injected into the body, mainly as ammonia, within 12 h. Like T. lymma, it also excreted the injected ammonia mainly as ammonia during the first 3 h postinjection. During this period, the percentage of the injected ammonia excreted in fish exposed to brackish water (28.4%+/-4.6%) was significantly lower than those exposed to freshwater (56.1%+/-8.26%). In contrast, the percentage of nitrogenous wastes being excreted as urea in the former (38.4%) was significantly greater than that in the latter (14.1%). These results suggest that a portion of the ammonia injected into the fish was turned into urea, and urea synthesis was increased transiently in fish exposed to brackish water during the initial postinjection period. However, urea was not retained effectively by H. signifer. Taken together, these results suggest that the primary function of the ornithine-urea cycle in ureogenic marine and freshwater elasmobranchs is to synthesize urea for osmotic water retention and not for ammonia detoxification.

Changes in salinity and ionic compositions can act as environmental signals to induce a reduction in ammonia production in the African lungfish Protopterus dolloi.

The slender African lungfish, Protopterus dolloi, does not aestivate in a subterranean mud cocoon, but is capable of aestivating inside a layer of dried mucus on land during drought. In this study, we aimed to elucidate if a slight increase in salinity in association with changes in the ionic composition could act as signals for P. dolloi to decrease endogenous ammonia production, in preparation for aestivation when the external medium dries up. Specimens of P. dolloi exposed to 3 per thousand water for 6 days exhibited consistently lower daily urea excretion rate than the freshwater control. This led to significant decreases in the cumulative total nitrogenous wastes excreted on days 3, 5 and 6. On day 6, there were decreases in urea contents in various tissues and organs. Taken together, these results suggest that there was a decrease in the rate of urea synthesis, the magnitude of which was greater than the decrease in the rate of urea excretion, and therefore resulted in decreases in internal urea contents. A decrease in the rate of urea synthesis should result in a decrease in the rate of glutamine utilization, and subsequently led to the accumulations of glutamine and/or ammonia. However, there were no changes in contents of glutamine and ammonia in various tissues and organs in the experimental animals. A logical explanation for this is that there must be a simultaneous reduction in ammonia production; if not, ammonia would accumulate due to the decrease in rate of urea synthesis. Since fish were unfed during the experiment, endogenous ammonia must be derived mainly from amino acid catabolism. Therefore, these results suggest that a suppression of amino acid catabolism occurred in specimens exposed to 3 per thousand for 6 days. The differences in effects of freshwater and 3 per thousand water on endogenous ammonia production could not be due to food deprivation because both groups of fish were fasted for the same period. Because control and experimental fish were kept in water and because there were no changes in the wet mass of the fish and blood osmolality before and after the experiment, dehydration did not occur. Furthermore, both groups of fish have comparable blood pH, pO2 and pCO2 on day 6 as they had free access to air, and therefore CO2 retention could be eliminated as the initiating factor of suppressed endogenous ammonia production. In conclusion, our results suggest that P. dolloi could respond to increases in salinity and changes in ionic composition in the external medium by suppressing ammonia production in preparation for aestivation when the water dries up.

Effects of intra-peritoneal injection with NH4Cl, urea, or NH4Cl+urea on nitrogen excretion and metabolism in the African lungfish Protopterus dolloi.

This study aimed to (1) determine if ammonia (as NH(4)Cl) injected intra-peritoneally into the ureogenic slender African lungfish, Protopterus dolloi, was excreted directly rather than being converted to urea; (2) examine if injected urea was retained in this lungfish, leading to decreases in liver arginine and brain tryptophan levels, as observed during aestivation on land; and (3) elucidate if increase in internal ammonia level would affect urea excretion, when ammonia and urea are injected simultaneously into the fish. Despite being ureogenic, P. dolloi rapidly excreted the excess ammonia as ammonia within the subsequent 12 h after NH(4)Cl was injected into its peritoneal cavity. Injected ammonia was not detoxified into urea through the ornithine-urea cycle, probably because it is energetically intensive to synthesize urea and because food was withheld before and during the experiment. In addition, injected ammonia was likely to stay in extracellular compartments available for direct excretion. At hour 24, only a small amount of ammonia accumulated in the muscle of these fish. In contrast, when urea was injected intra-peritoneally into P. dolloi, only a small percentage (34%) of it was excreted during the subsequent 24-h period. A significant increase in the rate of urea excretion was observed only after 16 h. At hour 24, significant quantities of urea were retained in various tissues of P. dolloi. Injection with urea led to an apparent reduction in endogenous ammonia production, a significant decrease in the hepatic arginine content, and a significantly lower level of brain tryptophan in this lungfish. All three phenomena had been observed previously in aestivating P. dolloi. Hence, it is logical to deduce that urea synthesis and accumulation could be one of the essential factors in initiating and perpetuating aestivation in this lungfish. Through the injection of NH(4)Cl + urea, it was demonstrated that an increase in urea excretion occurred in P. dolloi within the first 12 h post-injection, which was much earlier than that of fish injected with urea alone. These results suggest that urea excretion in P. dolloi is likely to be regulated by the level of internal ammonia in its body.

Nitrogen metabolism in the African lungfish (Protopterus dolloi) aestivating in a mucus cocoon on land.

This study aimed to elucidate the strategies adopted by the African slender lungfish, Protopterus dolloi, to ameliorate the toxicity of ammonia during short (6 days) or long (40 days) periods of aestivation in a layer of dried mucus in open air in the laboratory. Despite decreases in rates of ammonia and urea excretion, the ammonia content in the muscle, liver, brain and gut of P. dolloi remained unchanged after 6 days of aestivation compared with the control fasted for 6 days. For specimens aestivated for 40 days, the ammonia contents in the muscle, liver and gut were significantly lower than those of the control fasted for 40 days, which suggests a decrease in the rate of ammonia production. In addition, there were significant increases in contents of alanine, aspartate and glutamate in the muscle, which suggests decreases in their catabolism. During the first 6 days and the last 34 days of aestivation, the rate of ammonia production was reduced to 26% and 28%, respectively, of the control rate (6.83 micromol day(-1) g(-1) on day 0). During the first 6 days and the next 34 days of aestivation, the averaged urea synthesis rate was 2.39-fold and 3.8-fold, respectively, greater than the value of 0.25 micromol day(-1) g(-1) for the day 0 control kept in water. No induction of activities of the ornithine-urea cycle (OUC) enzymes was observed in specimens aestivated for 6 days, because the suppression of ammonia production led to a light demand on the OUC capacity. For specimens aestivated for 40 days, the activities of carbamoyl phosphate synthetase, ornithine transcarbamylase and argininosuccinate synthetase + lyase were significantly greater than those of the control fasted for 40 days. This is in agreement with the observation that the rate of urea synthesis in the last 34 days was greater than that in the first 6 days of aestivation. P. dolloi aestivated in a thin layer of dried mucus in open air with high O(2) tension throughout the 40 days of aestivation, which could be the reason why it was able to sustain a high rate of urea synthesis despite this being an energy-intensive process. Our results indicate that a reduction in ammonia production and decreases in hepatic arginine and cranial tryptophan contents are important facets of aestivation in P. dolloi.

Urea synthesis in the African lungfish Protopterus dolloi--hepatic carbamoyl phosphate synthetase III and glutamine synthetase are upregulated by 6 days of aerial exposure.

Like the marine ray Taeniura lymma, the African lungfish Protopterus dolloi possesses carbamoyl phosphate III (CPS III) in the liver and not carbamoyl phosphate I (CPS I), as in the mouse Mus musculus or as in other African lungfish reported elsewhere. However, similar to other African lungfish and tetrapods, hepatic arginase of P. dolloi is present mainly in the cytosol. Glutamine synthetase activity is present in both the mitochondrial and cytosolic fractions of the liver of P. dolloi. Therefore, we conclude that P. dolloi is a more primitive extant lungfish, which is intermediate between aquatic fish and terrestrial tetrapods, and represents a link in the fish-tetrapod continuum. During 6 days of aerial exposure, the ammonia excretion rate in P. dolloi decreased significantly to 8-16% of the submerged control. However, there were no significant increases in ammonia contents in the muscle, liver or plasma of specimens exposed to air for 6 days. These results suggest that (1). endogenous ammonia production was drastically reduced and (2). endogenous ammonia was detoxified effectively into urea. Indeed, there were significant decreases in glutamate, glutamine and lysine levels in the livers of fish exposed to air, which led to a decrease in the total free amino acid content. This indirectly confirms that the specimen had reduced its rates of proteolysis and/or amino acid catabolism to suppress endogenous ammonia production. Simultaneously, there were significant increases in urea levels in the muscle (8-fold), liver (10.5-fold) and plasma (12.6-fold) of specimens exposed to air for 6 days. Furthermore, there was an increase in the hepatic ornithine-urea cycle (OUC) capacity, with significant increases in the activities of CPS III (3.8-fold), argininosuccinate synthetase + lyase (1.8-fold) and, more importantly, glutamine synthetase (2.2-fold). This is the first report on the upregulation of OUC capacity and urea synthesis rate in an African lungfish exposed to air. Upon re-immersion, the urea excretion rate increased 22-fold compared with that of the control specimen, which is the greatest increase among fish during emersion-immersion transitions and suggests that P. dolloi possesses transporters that facilitate the excretion of urea in water.

A comparison of the effects of environmental ammonia exposure on the Asian freshwater stingray Himantura signifer and the Amazonian freshwater stingray Potamotrygon motoro.

The white-edge whip tail ray Himantura signifer inhabits a freshwater environment but has retained the capability to synthesize urea de novo through the arginine-ornithine-urea cycle (OUC). The present study aimed to elucidate whether the capacity of urea synthesis in H. signifer could be upregulated in response to environmental ammonia exposure. When H. signifer was exposed to environmental ammonia, fairly high concentrations of ammonia were accumulated in the plasma and other tissues. This would subsequently reduce the net influx of exogenous ammonia by reducing the NH(3) partial pressure gradient across the branchial and body surfaces. There was also an increase in the OUC capacity in the liver. Since the ammonia produced endogenously could not be excreted effectively in the presence of environmental ammonia, it was detoxified into urea through the OUC. In comparison, the South American freshwater stingray Potamotrygon motoro, which has lost the capability to synthesize urea de novo, was unable to detoxify ammonia to urea during ammonia loading. No increase in glutamine was observed in the various tissues of H. signifer exposed to environmental ammonia despite a significant increase in the hepatic glutamine synthetase activity. These results indicate that the excess glutamine formed was channelled completely into urea formation through carbamoyl phosphate synthetase III. It has been reported elsewhere that both urea synthesis and urea retention were upregulated in H. signifer exposed to 20 per thousand water for osmoregulatory purposes. By contrast, for H. signifer exposed to environmental ammonia in freshwater, the excess urea formed was excreted to the external medium instead. This suggests that the effectiveness of urea synthesis de novo as a strategy to detoxify ammonia is determined not simply by an increase in the capacity of urea synthesis but, more importantly, by the ability of the animal to control the direction (i.e. absorption or excretion) and rate of urea transport. Our results suggest that such a strategy began to develop in those elasmobranchs, e.g. H. signifer, that migrate into a freshwater environment from the sea but not in those permanently adapted to a freshwater environment.

The osmotic response of the Asian freshwater stingray (Himantura signifer) to increased salinity: a comparison with marine (Taeniura lymma) and Amazonian freshwater (Potamotrygon motoro) stingrays.

The white-edge freshwater whip ray Himantura signifer can survive in freshwater (0.7 per thousand ) indefinitely or in brackish water (20 per thousand ) for at least two weeks in the laboratory. In freshwater, the blood plasma was maintained hyperosmotic to that of the external medium. There was approximately 44 mmol l(-1) of urea in the plasma, with the rest of the osmolality made up mainly by Na(+) and Cl(-). In freshwater, it was not completely ureotelic, excreting up to 45% of its nitrogenous waste as urea. Unlike the South American freshwater stingray Potamotrygon motoro, H. signifer has a functional ornithine-urea cycle (OUC) in the liver, with hepatic carbamoylphosphate synthetase III (CPS III) and glutamine synthetase (GS) activities lower than those of the marine blue-spotted fan tail ray Taeniura lymma. More importantly, the stomach of H. signifer also possesses a functional OUC, the capacity (based on CPS III activity) of which was approximately 70% that in the liver. When H. signifer was exposed to a progressive increase in salinity through an 8-day period, there was a continuous decrease in the rate of ammonia excretion. In 20 per thousand water, urea levels in the muscle, brain and plasma increased significantly. In the plasma, osmolality increased to 571 mosmol kg(-1), in which urea contributed 83 mmol l(-1). Approximately 59% of the excess urea accumulated in the tissues of the specimens exposed to 20 per thousand water was equivalent to the deficit in ammonia excretion through the 8-day period, indicating that an increase in the rate of urea synthesis de novo at higher salinities would have occurred. Indeed, there was an induction in the activity of CPS III in both the liver and stomach, and activities of GS, ornithine transcarbamoylase and arginase in the liver. Furthermore, there was a significant decrease in the rate of urea excretion during passage through 5 per thousand, 10 per thousand and 15 per thousand water. Although the local T. lymma in full-strength sea water (30 per thousand ) had a much greater plasma urea concentration (380 mmol l(-1)), its urea excretion rate (4.7 micromol day(-1) g(-1)) was comparable with that of H. signifier in 20 per thousand water. Therefore, H. signifer appears to have reduced its capacity to retain urea in order to survive in the freshwater environment and, consequently, it could not survive well in full-strength seawater.

The snakehead Channa asiatica accumulates alanine during aerial exposure, but is incapable of sustaining locomotory activities on land through partial amino acid catabolism.

The freshwater snakehead Channa asiatica is an obligatory air-breather that resides in slow-flowing streams and in crevices near riverbanks in Southern China. In its natural habitat, it may encounter bouts of aerial exposure during the dry seasons. In the laboratory, the ammonia excretion rate of C. asiatica exposed to terrestrial conditions in a 12 h:12 h dark:light regime was one quarter that of the submerged control. Consequently, the ammonia contents in the muscle, liver and plasma increased significantly, and C. asiatica was able to tolerate quite high levels of ammonia in its tissues. Urea was not the major product of ammonia detoxification in C. asiatica, which apparently did not possess a functioning ornithine urea cycle. Rather, alanine increased fourfold to 12.6 micromol g(-1) in the muscle after 48 h of aerial exposure. This is the highest level known in adult teleosts exposed to air or an ammonia-loading situation. The accumulated alanine could account for 70% of the deficit in ammonia excretion during this period, indicating that partial amino acid catabolism had occurred. This would allow the utilization of certain amino acids as energy sources and, at the same time, maintain the new steady state levels of ammonia in various tissues, preventing them from rising further. There was a reduction in the aminating activity of glutamate dehydrogenase from the muscle and liver of specimens exposed to terrestrial conditions. Such a phenomenon has not been reported before and could, presumably, facilitate the entry of alpha-ketoglutarate into the Krebs cycle instead of its amination to glutamate, as has been suggested elsewhere. However, in contrast to mudskippers, C. asiatica was apparently unable to reduce the rates of proteolysis and amino acid catabolism, because the reduction in nitrogenous excretion during 48 h of aerial exposure was completely balanced by nitrogenous accumulation in the body. Alanine accumulation also occurred in specimens exposed to terrestrial conditions in total darkness, with no change in the total free amino acid content in the muscle. Exercise on land led to a decrease in glycogen content, and an increase in lactate levels, with no significant effect on ammonia and alanine contents in the muscle of C. asiatica. Hence, unlike the mudskipper Periophthalmodon schlosseri, C. asiatica was incapable of increasing the rate of partial amino acid catabolism to sustain locomotory activities on land. Alanine formation therefore appears to be a common strategy adopted by obligatory air-breathing fishes to avoid ammonia toxicity (not a strategy to detoxify ammonia) on land, but not all of them can utilize it to fuel muscular activities.