Axin1 and Axin2 regulate the WNT-signaling landscape to promote distinct mesoderm programs.

How distinct mesodermal lineages - extraembryonic, lateral, intermediate, paraxial and axial - are specified from pluripotent epiblast during gastrulation is a longstanding open question. By investigating AXIN, a negative regulator of the WNT/ß-catenin pathway, we have uncovered new roles for WNT signaling in the determination of mesodermal fates. We undertook complementary approaches to dissect the role of WNT signaling that augmented a detailed analysis of Axin1 ; Axin2 mutant mouse embryos, including single-cell and single-embryo transcriptomics, with in vitro pluripotent Epiblast-Like Cell differentiation assays. This strategy allowed us to reveal two layers of regulation. First, WNT initiates differentiation of primitive streak cells into mesoderm progenitors, and thereafter, WNT amplifies and cooperates with BMP/pSMAD1/5/9 or NODAL/pSMAD2/3 to propel differentiating mesoderm progenitors into either posterior streak derivatives or anterior streak derivatives, respectively. We propose that Axin1 and Axin2 prevent aberrant differentiation of pluripotent epiblast cells into mesoderm by spatially and temporally regulating WNT signaling levels.

Tracking early mammalian organogenesis - prediction and validation of differentiation trajectories at whole organism scale.

Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-h intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled timecourse of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-seq to transcriptionally determine cell fates of grafted primitive streak regions after 24 h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair.

A degron-based approach to manipulate Eomes functions in the context of the developing mouse embryo.

The T-box transcription factor Eomesodermin (Eomes), also known as Tbr2, plays essential roles in the early mouse embryo. Loss-of-function mutant embryos arrest at implantation due to Eomes requirements in the trophectoderm cell lineage. Slightly later, expression in the visceral endoderm promotes anterior visceral endoderm formation and anterior-posterior axis specification. Early induction in the epiblast beginning at day 6 is necessary for nascent mesoderm to undergo epithelial to mesenchymal transition (EMT). Eomes acts in a temporally and spatially restricted manner to sequentially specify the yolk sac haemogenic endothelium, cardiac mesoderm, definitive endoderm, and axial mesoderm progenitors during gastrulation. Little is known about the underlying molecular mechanisms governing Eomes actions during the formation of these distinct progenitor cell populations. Here, we introduced a degron-tag and mCherry reporter sequence into the Eomes locus. Our experiments analyzing homozygously tagged embryonic stem cells and embryos demonstrate that the degron-tagged Eomes protein is fully functional. dTAG (degradation fusion tag) treatment in vitro results in rapid protein degradation and recapitulates the Eomes-null phenotype. However in utero administration of dTAG resulted in variable and lineage-specific degradation, likely reflecting diverse cell type-specific Eomes expression dynamics. Finally, we demonstrate that Eomes protein rapidly recovers following dTAG wash-out in vitro. The ability to temporally manipulate Eomes protein expression in combination with cell marking by the mCherry-reporter offers a powerful tool for dissecting Eomes-dependent functional roles in these diverse cell types in the early embryo.

The T-box transcription factor Eomesodermin governs haemogenic competence of yolk sac mesodermal progenitors.

Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM. Eomes regulates the accessibility of enhancers that the transcription factor stem cell leukaemia (SCL) normally utilizes to specify primitive erythrocytes and is essential for the normal development of Runx1+ haemogenic endothelium. Single-cell RNA sequencing suggests that Eomes loss of function profoundly blocks the formation of blood progenitors but not specification of Flk-1+ haematoendothelial progenitors. Our findings place Eomes at the top of the transcriptional hierarchy regulating early blood formation and suggest that haemogenic competence is endowed earlier during embryonic development than was previously appreciated.

Comorbidity and prognosis in head and neck cancers: Differences by subsite, stage, and human papillomavirus status.

BACKGROUND: The prognostic utility of comorbidity on head and neck cancer may differ by subsite, stage, and human papillomavirus (HPV) status. METHODS: We reviewed the medical records of 4953 patients with head and neck cancer for comorbidity (Charlson Comorbidity Index [CCI]), smoking, and alcohol history. Multivariate proportional hazards assessed the association of CCI with survival. HPV status was determined using p16 immunohistochemistry. RESULTS: After accounting for stage, higher CCI was associated with worse overall survival (OS) in nasopharyngeal (hazard ratio [HR], 2.93; 95% confidence interval [CI], 1.53-5.62), oropharyngeal (HR, 1.99; 95% CI, 1.63-2.43), and oral cavity cancers (HR, 1.54; 95% CI, 1.27-1.86). These associations were most prominent in the early stage oral cavity (HR, 2.11; 95% CI, 1.50-2.96) and laryngeal (HR, 1.87; 95% CI, 1.35-2.58) cancers, and in advanced stage oropharyngeal (HR, 2.23; 95% CI, 1.81-2.74) and nasopharyngeal (HR, 3.50; 95% CI, 1.76-6.97) cancers. CCI was independently prognostic even in the HPV-adjusted oropharyngeal cancers. CONCLUSION: Comorbidity was prognostic in subsets of nasopharyngeal, oropharyngeal, oral cavity, and laryngeal cancers. Comorbidity may be a partial surrogate for age and social habits.

The changing incidence of human papillomavirus-associated oropharyngeal cancer using multiple imputation from 2000 to 2010 at a Comprehensive Cancer Centre.

INTRODUCTION: Human papillomavirus (HPV) is a risk and prognostic factor for oropharyngeal cancer (OPC). Determining whether the incidence of HPV-associated OPC is rising informs health policy. METHODS: HPV status was ascribed using p16 immunohistochemistry in 683/1474 OPC patients identified from the Princess Margaret Hospital's Cancer Registry (from 2000 to 2010). Missing p16 data was estimated using multiple (n=100) imputation (MI) and validated using an independent OPC cohort (n=214). Non-OPC head and neck squamous cell carcinoma (HNSCC) (n=3262) were also used for time-trend comparison. Regression was used to compare HNSCC subsets and time-trends. The c-index was used to measure the predictive ability of MI. RESULTS: The incidence of OPC rose from 23.3% of all HNSCC in 2000 to 31.2% in 2010 (p=0.002). In the subset of OPC tested for p16, there was no change in p16 positivity over time (p=0.9). However, p16 testing became more frequent over time (p<0.0001), but was nonetheless biased, favouring never-smokers [OR 1.87 (95% CI 1.29-2.70)] and tumors of the tonsil [OR 2.30 (1.52-3.47)] or base-of-tongue [OR 1.72 (1.10-2.70)]. These same factors were also associated with p16-positivity [ORs 3.22 (1.27-8.16), 7.26 (3.50-15.1), 5.83 (2.70-12.7), respectively]. Following MI and normalization, the proportion of OPC that was p16-associated rose from 39.8% in 2000 to 65.0% in 2010, p=0.002, fully explaining the rise in OPC in our patient population. CONCLUSION: The rise in HNSCC referrals seen from 2000 to 2010 at our institution was driven primarily by p16-associated OPC. MI was necessary to derive reliable conclusions when cases with missing data are considerable.

Validation of a one-page patient-reported Charlson comorbidity index questionnaire for upper aerodigestive tract cancer patients.

OBJECTIVES: Cancer patients have a wide range of comorbidities that are important confounders for biomarker and clinical studies of prognosis and outcome. Comorbidities can be captured using the Charlson Comorbidity Index (CCI) through abstraction of medical records, but patient-reported outcome (PRO) questionnaires have also been used. The objective was to validate the PRO-CCI in a head and neck cancer (HNC) population, and to assess its level of agreement with the standard (std-CCI) method of chart review. METHODS: A one-page PRO-CCI was compared with the std-CCI obtained through independent abstraction in 882 HNC patients (2007-2010). Kappa statistics and associated measures (p(pos) and p(neg)) were used to assess agreement. Discrepancy for each comorbid illness was evaluated. Proportional hazard models compared the association of std-CCI and PRO-CCI with overall survival (OS). Adjustments were made and a modified PRO-CCI was re-evaluated in a new cohort of upper aerodigestive tract cancers patient. RESULTS: PRO-CCI was higher than the std-CCI (p < 0.0001). After adjustment, having at least two comorbidities according to either the std-CCI [HR 1.97 (1.38-2.80)] or the PRO-CCI [HR 1.62 (1.18-2.24)] was prognostic. Of the most prevalent comorbidities, agreement was high for most of the CCI elements (kappa 0.76-0.93), but poorest agreement for connective tissue disease (kappa = 0.29, p(pos) = 43%, p(neg) = 84%) and COPD (kappa = 0.48, p(pos) = 53%, p(neg) = 95%). When the connective tissue disease question was modified, agreement of this item improved (kappa = 0.47, p(pos) = 50%). CONCLUSION: PRO-CCI can be an easy and effective tool in prognostic and outcomes research in HNC patients.