Lineage tracing reveals the phylodynamics, plasticity, and paths of tumor evolution.

Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression.

Avian red blood cell mitochondria produce more heat in winter than in autumn.

Endotherms in cold regions improve heat-producing capacity when preparing for winter. We know comparatively little about how this change is fueled by seasonal adaptation in cellular respiration. Thus, we studied the changes of mitochondrial function in red blood cells in sympatric Coal (Periparus ater), Blue (Cyanistes caeruleus), and Great (Parus major) tits between autumn and winter. These species differ more than twofold in body mass and in several aspects of their foraging ecology and social dominance, which could require differential seasonal adaptation of energy expenditure. Coal and Great tits in particular upregulated the mitochondrial respiration rate and mitochondrial volume in winter. This was not directed toward ATP synthesis, instead reflecting increased uncoupling of electron transport from ATP production. Because uncoupling is exothermic, this increased heat-producing capacity at the sub-cellular level in winter. This previously unexplored the route of thermogenesis in birds should be addressed in future work.

Experimental warming during incubation improves cold tolerance of blue tit (Cyanistes caeruleus) chicks.

Climate change and increasing air temperature may alter environmental conditions for developing birds, with a range of phenotypic consequences for offspring. The thermal environment during incubation may affect the trade-off between growth and thermoregulation, but the effects of temperature on the ontogeny of endothermy are not fully understood. Therefore, we experimentally tested whether heating the nest cup of Eurasian blue tits (Cyanistes caeruleus) during incubation would influence cold tolerance of the chicks after hatching. Chicks from both heated and control nests showed a decrease in cooling rate with age as they became increasingly endothermic and homeothermic. However, chicks from previously heated nests cooled at a lower rate per unit surface area and from across the whole body. These chicks also had a greater body mass during the first 12 days of life compared with chicks from control nests. Lower cooling rates in heated chicks may reflect greater thermogenic capacity or a reduced surface area to volume ratio owing to a greater body mass. Future projections for climate change predict rising air temperature and increased likelihood of heatwaves, even in temperate regions. Our results indicate that nest microclimate can affect thermoregulation in offspring, and thus may be used to predict some of the future physiological responses of birds to climate change during breeding.

Transgenic overexpression of ribonucleotide reductase improves cardiac performance.

We previously demonstrated that cardiac myosin can use 2-deoxy-ATP (dATP) as an energy substrate, that it enhances contraction and relaxation with minimal effect on calcium-handling properties in vitro, and that contractile enhancement occurs with only minor elevation of cellular [dATP]. Here, we report the effect of chronically enhanced dATP concentration on cardiac function using a transgenic mouse that overexpresses the enzyme ribonucleotide reductase (TgRR), which catalyzes the rate-limiting step in de novo deoxyribonucleotide biosynthesis. Hearts from TgRR mice had elevated left ventricular systolic function compared with wild-type (WT) mice, both in vivo and in vitro, without signs of hypertrophy or altered diastolic function. Isolated cardiomyocytes from TgRR mice had enhanced contraction and relaxation, with no change in Ca(2+) transients, suggesting targeted improvement of myofilament function. TgRR hearts had normal ATP and only slightly decreased phosphocreatine levels by (31)P NMR spectroscopy, and they maintained rate responsiveness to dobutamine challenge. These data demonstrate long-term (at least 5-mo) elevation of cardiac [dATP] results in sustained elevation of basal left ventricular performance, with maintained ß-adrenergic responsiveness and energetic reserves. Combined with results from previous studies, we conclude that this occurs primarily via enhanced myofilament activation and contraction, with similar or faster ability to relax. The data are sufficiently compelling to consider elevated cardiac [dATP] as a therapeutic option to treat systolic dysfunction.

Ribonucleotide reductase is not limiting for mitochondrial DNA copy number in mice.

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in deoxyribonucleoside triphosphate (dNTP) biosynthesis, with important roles in nuclear genome maintenance. RNR is also essential for maintenance of mitochondrial DNA (mtDNA) in mammals. The mechanisms regulating mtDNA copy number in mammals are only being discovered. In budding yeast, RNR overexpression resulted in increased mtDNA levels, and rescued the disease phenotypes caused by a mutant mtDNA polymerase. This raised the question of whether mtDNA copy number increase by RNR induction could be a strategy for treating diseases with mtDNA mutations. We show here that high-level overexpression of RNR subunits (Rrm1, Rrm2 and p53R2; separately or in different combinations) in mice does not result in mtDNA copy number elevation. Instead, simultaneous expression of two RNR subunits leads to imbalanced dNTP pools and progressive mtDNA depletion in the skeletal muscle, without mtDNA mutagenesis. We also show that endogenous RNR transcripts are downregulated in response to large increases of mtDNA in mice, which is indicative of nuclear-mitochondrial crosstalk with regard to mtDNA copy number. Our results establish that RNR is not limiting for mtDNA copy number in mice, and provide new evidence for the importance of balanced dNTP pools in mtDNA maintenance in postmitotic tissues.

Mice lacking FABP9/PERF15 develop sperm head abnormalities but are fertile.

The male germ cell-specific fatty acid-binding protein 9 (FABP9/PERF15) is the major component of the murine sperm perforatorium and perinuclear theca. Based on its cytoskeletal association and sequence homology to myelin P2 (FABP8), it has been suggested that FABP9 tethers sperm membranes to the underlying cytoskeleton. Furthermore, its upregulation in apoptotic testicular germ cells and its increased phosphorylation status during capacitation suggested multiple important functions for FABP9. Therefore, we investigated specific functions for FABP9 by means of targeted gene disruption in mice. FABP9(-/-) mice were viable and fertile. Phenotypic analysis showed that FABP9(-/-) mice had significant increases in sperm head abnormalities (~8% greater than their WT cohorts); in particular, we observed the reduction or absence of the characteristic structural element known as the "ventral spur" in ~10% of FABP9(-/-) sperm. However, deficiency of FABP9 affected neither membrane tethering to the perinuclear theca nor the fatty acid composition of sperm. Moreover, epididymal sperm numbers were not affected in FABP9(-/-) mice. Therefore, we conclude that FABP9 plays only a minor role in providing the murine sperm head its characteristic shape and is not absolutely required for spermatogenesis or sperm function.

Getting ahead: context-dependent responses to odorant filaments drive along-stream progress during odor tracking in blue crabs.

The chemosensory signal structure governing the upstream progress of blue crabs to an odorant source was examined. We used a three-dimensional laser-induced fluorescence system to collect chemical concentration data simultaneously with behavior observations of actively tracking blue crabs (Callinectes sapidus) in a variety of plume types. This allowed us to directly link chemical signal properties at the antennules and legs to subsequent upstream motion while altering the spatial and temporal intermittency characteristics of the sensory field. Our results suggest that odorant stimuli elicit responses in a binary fashion by causing upstream motion, provided the concentration at the antennules exceeds a specific threshold. In particular, we observed a significant association between crab velocity changes and odorant spike encounters defined using a threshold that is scaled to the mean of the instantaneous maximum concentration. Thresholds were different for each crab, indicating a context-sensitive response to signal dynamics. Our data also indicate that high frequency of odorant spike encounters terminate upstream movement. Further, the data provide evidence that the previous state of the crab and prior stimulus history influence the behavioral response (i.e. the response is context dependent). Two examples are: (1) crabs receiving prior odorant spikes attained elevated velocity more quickly in response to subsequent spikes; and (2) prior acceleration or deceleration of the crab influenced the response time period to a particular odorant spike. Finally, information from both leg and antennule chemosensors interact, suggesting parallel processing of odorant spike properties during navigation.

Staying the course: chemical signal spatial properties and concentration mediate cross-stream motion in turbulent plumes.

This study examined the role of broadly distributed sensor populations in chemosensory searching, especially cross-stream heading adjustment. We used three-dimensional laser-induced fluorescence to collect chemical concentration data simultaneously with behavior observations of actively tracking blue crabs (Callinectes sapidus). Our analysis indicates that the spatial distribution of the odorant concentration field is necessary and sufficient to mediate correct cross-stream motion, although concentration provides information that supplements that obtained from the spatial distribution. Crab movement is continually adjusted to maintain an upstream heading, with corrections toward the source modulated only in the presence of chemical cues. Crabs detect and respond to shifts in the position of the center-of-mass (COM) of the odorant concentration distribution as small as 5% of the leg span, which corresponds to ∼0.8-0.9 cm. The reaction time after a 5% threshold shift in the position of the COM is in the range of 2-4 s. Data also indicate that these steering responses are dependent on stimulus history or other characteristics of the plume, with crabs taking longer to respond in conditions with large-scale spatial meanders. Although cross-stream motion is determined by chemical signal inputs to receptors on the walking legs, crabs do make rotational movements in response to chemical signals impinging on the antennules. These rotational movements do not affect the direction of travel, but rather, determine the crab's body angle with respect to the flow. Interestingly, these body angles seem to represent a compromise between reducing drag and obtaining better chemical signal information, and this trade-off is resolved differently under different plume conditions.

An ectopic expression screen reveals the protective and toxic effects of Drosophila seminal fluid proteins.

In Drosophila melanogaster, seminal fluid regulates the reproductive and immune responses of mated females. Some seminal fluid proteins may provide protective functions to mated females, such as antimicrobial activity and/or stimulation of antimicrobial gene expression levels, while others appear to have negative effects, contributing to a "cost of mating." To identify seminal proteins that could participate in these phenomena, we used a systemic ectopic expression screen to test the effects on unmated females of proteins normally produced by the male accessory gland (Acps). Of the 21 ectopically expressed Acps that we tested for ability to assist in the clearance of a bacterial infection with Serratia marcescens, 3 Acps significantly reduced the bacterial counts of infected females, suggesting a protective role. Of the 23 Acps that we tested for toxicity, 3 were toxic, including one that has been implicated in the cost of mating in another study. We also tested ectopic expression females for other Acp-induced effects, but found no additional Acps that affected egg laying or receptivity upon ectopic expression.

Broad overexpression of ribonucleotide reductase genes in mice specifically induces lung neoplasms.

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in nucleotide biosynthesis and plays a central role in genome maintenance. Although a number of regulatory mechanisms govern RNR activity, the physiologic effect of RNR deregulation had not previously been examined in an animal model. We show here that overexpression of the small RNR subunit potently and selectively induces lung neoplasms in transgenic mice and is mutagenic in cultured cells. Combining RNR deregulation with defects in DNA mismatch repair, the cellular mutation correction system, synergistically increased RNR-induced mutagenesis and carcinogenesis. Moreover, the proto-oncogene K-ras was identified as a frequent mutational target in RNR-induced lung neoplasms. Together, these results show that RNR deregulation promotes lung carcinogenesis through a mutagenic mechanism and establish a new oncogenic activity for a key regulator of nucleotide metabolism. Importantly, RNR-induced lung neoplasms histopathologically resemble human papillary adenocarcinomas and arise stochastically via a mutagenic mechanism, making RNR transgenic mice a valuable model for lung cancer.