A novel Yap/Taz zebrafish reporter reveals a role of Hippo pathway transducers in angiogenesis

YAP and TAZ, by orchestrating cell proliferation, cell death and cell-fate decisions, are key players of a complex network of signaling pathways acting during development. Deregulation of YAP/TAZ signaling causes robust organ overgrowth during organogenesis, which translates to loss of tissue homeostasis in the adult and consequent cancer development. YAP/TAZ are transcriptional co-activators that interact with TEAD transcription factors to promote cell proliferation and survival. Their transcriptional activity is regulated by nucleocytoplasmic shuttling and nuclear accumulation, which are controlled by the Hippo kinase cascade, but also by mechanical cues sensed by the cell and by other pathways. Among these, Wnt/β-catenin takes on a particular relevance, since it was recently shown to regulate YAP/TAZ activity through AXIN-mediated sequestration of YAP/TAZ in the β-catenin destruction complex. Here, we describe the generation, validation and characterization of a novel biosensor zebrafish reporting the activity of Yap/Taz. It expresses nuclear mCherry, eGFP or the destabilized green fluorescent protein VenusPEST under the control of a promoter fragment of the human YAP/TAZ target gene CTGF, that contains 3 TEAD DNA-binding sites. Several independent founder fish transmitting the transgene to the germline were identified and used to establish the stable reporter lines. All stable transgenic fish shared a similar expression pattern, which was maintained in subsequent generations. Knockdown and overexpression approaches were used to validate the reporter. Co-injection of two morpholinos targeting Yap and Taz pre-mRNAs reduced the reporter signal, whereas injection of mRNAs coding for a constitutively active form of Yap, Taz and Tead (YAP-5SA, TAZ-4SA, TEAD-VP16) increased it. The CTGF-based transgenic lines represent therefore bona fide Yap/Taz reporters. During development, strong reporter signal is visible mainly in the lens and otic vesicles, the pharyngeal arches, the heart, the pectoral fin and the vasculature, but the reporter protein expression is also detected in many other tissues and organs. The almost ubiquitous activation of Yap/Taz observed during early embryogenesis, consistent with the general role of YAP/TAZ in promoting cell proliferation and organ growth, is largely silenced in the adult fish, where the reporter signal is restricted to the lens, the ovary, the heart and the whole vasculature. We also showed that the CTGF-based biosensor zebrafish is able to report Yap/Taz activation during larval and adult fin regeneration, as expected from the role that YAP/TAZ signaling plays in the regenerative processes. The zebrafish CTGF-based reporter permitted to show in a living organism during development the regulation that the Wnt/β-catenin pathway exerts on Yap/Taz activity. Our results in terms of variations of the reporter signal, after both genetic and pharmacological modulation of the Wnt pathway activity, are in accordance with the model recently depicted in vitro. The general and sustained reporter activity we observed in the endothelium during embryogenesis suggested a functional involvement of Yap/Taz signaling in developmental angiogenesis. Yap/Taz knockdown impaired the intersegmental vessels (ISVs) growth, while the overactivation of Yap/Taz-mediated transcription caused an aberrant sprouting from the ISVs. The vessel sprouting-promoting capacity of Yap/Taz is cell-autonomous, as the same phenomenon was observed by expressing TAZ-4SA under the control of an endothelium-specific promoter. The CTGF-based zebrafish reporter is a new powerful tool to study in vivo Yap/Taz pathway activation, with possible applications in drug screening, regeneration and cancer biology. It permitted to confirm in vivo during development the crosstalk between Wnt/β-catenin and Yap/Taz pathways and to discover a novel role of Yap/Taz in vessel sprouting, suggesting a pro-angiogenic function of YAP/TAZ transcriptional activity

Zebrafish mutants and TEAD reporters reveal essential functions for Yap and Taz in posterior cardinal vein development

As effectors of the Hippo signaling cascade, YAP1 and TAZ are transcriptional regulators playing important roles in development, tissue homeostasis and cancer. A number of different cues, including mechanotransduction of extracellular stimuli, adhesion molecules, oncogenic signaling and metabolism modulate YAP1/TAZ nucleo-cytoplasmic shuttling. In the nucleus, YAP1/TAZ tether with the DNA binding proteins TEADs, to activate the expression of target genes that regulate proliferation, migration, cell plasticity, and cell fate. Based on responsive elements present in the human and zebrafish promoters of the YAP1/TAZ target gene CTGF, we established zebrafish fluorescent transgenic reporter lines of Yap1/Taz activity. These reporter lines provide an in vivo view of Yap1/Taz activity during development and adulthood at the whole organism level. Transgene expression was detected in many larval tissues including the otic vesicles, heart, pharyngeal arches, muscles and brain and is prominent in endothelial cells. Analysis of vascular development in yap1/taz zebrafish mutants revealed specific defects in posterior cardinal vein (PCV) formation, with altered expression of arterial/venous markers. The overactivation of Yap1/Taz in endothelial cells was sufficient to promote an aberrant vessel sprouting phenotype. Our findings confirm and extend the emerging role of Yap1/Taz in vascular development including angiogenesis

Adjuvant capecitabine in triple negative breast cancer patients with residual disease after neoadjuvant treatment: real-world evidence from CaRe, a multicentric, observational study

Background: In triple negative breast cancer patients treated with neoadjuvant chemotherapy, residual disease at surgery is the most relevant unfavorable prognostic factor. Current guidelines consider the use of adjuvant capecitabine, based on the results of the randomized CREATE-X study, carried out in Asian patients and including a small subset of triple negative tumors. Thus far, evidence on Caucasian patients is limited, and no real-world data are available. Methods: We carried out a multicenter, observational study, involving 44 oncologic centres. Triple negative breast cancer patients with residual disease, treated with adjuvant capecitabine from January 2017 through June 2021, were recruited. We primarily focused on treatment tolerability, with toxicity being reported as potential cause of treatment discontinuation. Secondarily, we assessed effectiveness in the overall study population and in a subset having a minimum follow-up of 2 years. Results: Overall, 270 patients were retrospectively identified. The 50.4% of the patients had residual node positive disease, 7.8% and 81.9% had large or G3 residual tumor, respectively, and 80.4% a Ki-67 >20%. Toxicity-related treatment discontinuation was observed only in 10.4% of the patients. In the whole population, at a median follow-up of 15 months, 2-year disease-free survival was 62%, 2 and 3-year overall survival 84.0% and 76.2%, respectively. In 129 patients with a median follow-up of 25 months, 2-year disease-free survival was 43.4%, 2 and 3-year overall survival 78.0% and 70.8%, respectively. Six or more cycles of capecitabine were associated with more favourable outcomes compared with less than six cycles. Conclusion: The CaRe study shows an unexpectedly good tolerance of adjuvant capecitabine in a real-world setting, although effectiveness appears to be lower than that observed in the CREATE-X study. Methodological differences between the two studies impose significant limits to comparability concerning effectiveness, and strongly invite further research

Supplement: "Localization and broadband follow-up of the gravitational-wave transient GW150914" (2016, ApJL, 826, L13)

This Supplement provides supporting material for Abbott et al. (2016a). We briefly summarize past electromagnetic (EM) follow-up efforts as well as the organization and policy of the current EM follow-up program. We compare the four probability sky maps produced for the gravitational-wave transient GW150914, and provide additional details of the EM follow-up observations that were performed in the different bands

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M>70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0<e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Ultralight vector dark matter search using data from the KAGRA O3GK run

Among the various candidates for dark matter (DM), ultralight vector DM can be probed by laser interferometric gravitational wave detectors through the measurement of oscillating length changes in the arm cavities. In this context, KAGRA has a unique feature due to differing compositions of its mirrors, enhancing the signal of vector DM in the length change in the auxiliary channels. Here we present the result of a search for U(1)B−L gauge boson DM using the KAGRA data from auxiliary length channels during the first joint observation run together with GEO600. By applying our search pipeline, which takes into account the stochastic nature of ultralight DM, upper bounds on the coupling strength between the U(1)B−L gauge boson and ordinary matter are obtained for a range of DM masses. While our constraints are less stringent than those derived from previous experiments, this study demonstrates the applicability of our method to the lower-mass vector DM search, which is made difficult in this measurement by the short observation time compared to the auto-correlation time scale of DM

Search for gravitational-lensing signatures in the full third observing run of the LIGO-Virgo network

Gravitational lensing by massive objects along the line of sight to the source causes distortions of gravitational wave-signals; such distortions may reveal information about fundamental physics, cosmology and astrophysics. In this work, we have extended the search for lensing signatures to all binary black hole events from the third observing run of the LIGO--Virgo network. We search for repeated signals from strong lensing by 1) performing targeted searches for subthreshold signals, 2) calculating the degree of overlap amongst the intrinsic parameters and sky location of pairs of signals, 3) comparing the similarities of the spectrograms amongst pairs of signals, and 4) performing dual-signal Bayesian analysis that takes into account selection effects and astrophysical knowledge. We also search for distortions to the gravitational waveform caused by 1) frequency-independent phase shifts in strongly lensed images, and 2) frequency-dependent modulation of the amplitude and phase due to point masses. None of these searches yields significant evidence for lensing. Finally, we use the non-detection of gravitational-wave lensing to constrain the lensing rate based on the latest merger-rate estimates and the fraction of dark matter composed of compact objects