Genome-wide RNA Tomography in the zebrafish embryo.

Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. Although genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using in situ hybridization, for example, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. Importantly, our technique enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.

Identification of a heterogeneous and dynamic ciliome during embryonic development and cell differentiation.

Primary cilia are nearly ubiquitous organelles that transduce molecular and mechanical signals. Although the basic structure of the cilium and the cadre of genes that contribute to ciliary formation and function (the ciliome) are believed to be evolutionarily conserved, the presentation of ciliopathies with narrow, tissue-specific phenotypes and distinct molecular readouts suggests that an unappreciated heterogeneity exists within this organelle. Here, we provide a searchable transcriptomic resource for a curated primary ciliome, detailing various subgroups of differentially expressed genes within the ciliome that display tissue and temporal specificity. Genes within the differentially expressed ciliome exhibited a lower level of functional constraint across species, suggesting organism and cell-specific function adaptation. The biological relevance of ciliary heterogeneity was functionally validated by using Cas9 gene-editing to disrupt ciliary genes that displayed dynamic gene expression profiles during osteogenic differentiation of multipotent neural crest cells. Collectively, this novel primary cilia-focused resource will allow researchers to explore longstanding questions related to how tissue and cell-type specific functions and ciliary heterogeneity may contribute to the range of phenotypes associated with ciliopathies.

COVID-19 Impacts on Primary Care Clinic Care Management Processes.

PURPOSE: To learn whether the COVID-19 pandemic's disruptions and associated reduced health outcomes for people with chronic conditions might have been caused by a decrease in care management processes (CMPs) in primary care clinics METHODS: Longitudinal cohort design with repeated survey-based measures of CMPs from 2017, 2019, and 2021 in 269 primary care clinics in Minnesota. RESULTS: There were only small differences in organizational characteristics and no differences in overall CMPs between the 269 clinics analyzed and the 287 that only completed surveys in 1 or 2 years. Overall CMP scores rose by similar amounts (1.6% and 2.1%) from 2017 to 2019 and from 2019 to 2021. In 2021, CMP scores were lower in small medical groups than in large medical groups in 2017 (66.1% vs 78.5%, P <.001), a similar difference to that in 2017. Care management process scores were also lower in clinics in urban areas compared with rural areas (73.9% vs 79.0%, P <.001), but overall scores in all subgroups were higher in 2021 than in 2017. This improvement occurred despite reports from 55% of clinic leaders that the pandemic had been very or extremely disruptive. CONCLUSIONS: Although quite disrupted by the pandemic, care management processes for chronic disease care in these resilient primary care clinics actually increased from 2019 to 2021, at least in clinics that were part of large organizations. However, that was not true for clinics from smaller groups and perhaps for other areas of care.

Successful Change Management Strategies for Improving Diabetes Care Delivery Among High-Performing Practices.

PURPOSE: To learn how the highest-performing primary care practices manage change when implementing improvements to diabetes care delivery. METHODS: We ranked a total of 330 primary care practices submitting practice management assessments and diabetes reports to the Understanding Infrastructure Transformation Effects on Diabetes study in 2017 and 2019 by Optimal Diabetes Care performance. We ranked practices from the top quartile by greatest annual improvement to capture dynamic change. Starting with the top performers, we interviewed practice leaders to identify their most effective strategies for managing change. Interview transcripts were qualitatively analyzed to identify change management strategies. Saturation occurred when no new strategies were identified over 2 consecutive interviews. RESULTS: Ten of the top 13 practices agreed to interviews. We identified 199 key comments representing 48 key care management concepts. We also categorized concepts into 6 care management themes and 37 strategic approaches. We categorized strategic approaches into 13 distinct change management strategies. The most common strategies identified were (1) standardizing the care process, (2) performance awareness, (3) enhancing care teams, (4) health care organization participation, (5) improving reporting systems, (6) engaging staff and clinicians, (7) accountability for tasks, (8) engaging leadership, and (9) tracking change. Care management themes identified by most practices included proactive care, improving patient relationships, and previsit planning. CONCLUSIONS: Top-performing primary care practices identify a similar group of strategies as important for managing change during quality improvement activities. Practices involved in diabetes improvement activities, and perhaps other chronic conditions, should consider adopting these change management strategies.

Corrigendum: High-throughput discovery of novel developmental phenotypes.

This corrects the article DOI: 10.1038/nature19356.

Genomic architecture of Shh-dependent cochlear morphogenesis.

The mammalian cochlea develops from a ventral outgrowth of the otic vesicle in response to Shh signaling. Mouse embryos lacking Shh or its essential signal transduction components display cochlear agenesis; however, a detailed understanding of the transcriptional network mediating this process is unclear. Here, we describe an integrated genomic approach to identify Shh-dependent genes and associated regulatory sequences that promote cochlear duct morphogenesis. A comparative transcriptome analysis of otic vesicles from mouse mutants exhibiting loss (Smoecko ) and gain (Shh-P1) of Shh signaling reveal a set of Shh-responsive genes partitioned into four expression categories in the ventral half of the otic vesicle. This target gene classification scheme provides novel insight into several unanticipated roles for Shh, including priming the cochlear epithelium for subsequent sensory development. We also mapped regions of open chromatin in the inner ear by ATAC-seq that, in combination with Gli2 ChIP-seq, identified inner ear enhancers in the vicinity of Shh-responsive genes. These datasets are useful entry points for deciphering Shh-dependent regulatory mechanisms involved in cochlear duct morphogenesis and establishment of its constituent cell types.

Progress towards completing the mutant mouse null resource.

The generation of a comprehensive catalog of null alleles covering all protein-coding genes is the goal of the International Mouse Phenotyping Consortium. Over the past 20 years, significant progress has been made towards achieving this goal through the combined efforts of many large-scale programs that built an embryonic stem cell resource to generate knockout mice and more recently employed CRISPR/Cas9-based mutagenesis to delete critical regions predicted to result in frameshift mutations, thus, ablating gene function. The IMPC initiative builds on prior and ongoing work by individual research groups creating gene knockouts in the mouse. Here, we analyze the collective efforts focusing on the combined null allele resource resulting from strains developed by the research community and large-scale production programs. Based upon this pooled analysis, we examine the remaining fraction of protein-coding genes focusing on clearly defined mouse-human orthologs as the highest priority for completing the mutant mouse null resource. In summary, we find that there are less than 3400 mouse-human orthologs remaining in the genome without a targeted null allele that can be further prioritized to achieve our overall goal of the complete functional annotation of the protein-coding portion of a mammalian genome.

High-throughput discovery of novel developmental phenotypes.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

Strategies and Factors Associated With Top Performance in Primary Care for Diabetes: Insights From a Mixed Methods Study.

PURPOSE: The aim of this study was to determine what strategies and factors are most important for high performance in the primary care of patients with diabetes. METHODS: We performed a mixed-methods, cross-sectional, observational analysis of interviews and characteristics of primary care clinics in Minnesota and bordering areas. We compared strategies, facilitators, and barriers identified by 31 leaders of 17 clinics in high-, middle-, and low-performance quartiles on a standardized composite measure of diabetes outcomes for 416 of 586 primary care clinics. Semistructured interview data were combined with quantitative data regarding clinic performance and a survey of the presence of care management processes. RESULTS: The interview analysis identified 10 themes providing unique insights into the factors and strategies characterizing the 3 performance groups. The main difference was the degree to which top-performing clinics used patient data to guide proactive and outreach methods to intensify treatment and monitor effect. Top clinics also appeared to view visit-based care management processes as necessary but insufficient, whereas all respondents regarded being part of a large system as mostly helpful. CONCLUSIONS: Top-performing clinic approaches to diabetes care differ from lower-performing clinics primarily by emphasizing data-driven proactive outreach to patients to intensify treatment. Although confirmatory studies are needed, clinical leaders should consider the value of this paradigm shift in approach to care.

Transcriptional regulatory control of mammalian nephron progenitors revealed by multi-factor cistromic analysis and genetic studies.

Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated 'regulatory hotspots' around genes closely associated with progenitor programs. To examine their functional significance, we deleted 'hotspot' enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis.

Differences in Diabetes Care With and Without Certification as a Medical Home.

PURPOSE: The purpose of this study was to assess whether primary care practices certified as medical homes differ in having the practice systems required for that designation and in attaining favorable outcomes for their patients with diabetes, and whether those systems are associated with better diabetes outcomes. METHODS: We undertook a cross-sectional observational study, Understanding Infrastructure Transformation Effects on Diabetes (UNITED), of 586 Minnesota adult primary care practices, comparing those that were certified vs uncertified as medical homes in 2017, with analyses supplemented by previously published studies of these practices. We collected survey information about the presence of medical home practice systems for diabetes care and obtained 6 standardized measures of diabetes care collected yearly from all Minnesota practices. RESULTS: Of 416 practices completing questionnaires (71% of all practices, 92% of participating practices), 394 had data on diabetes care measures. Uncertified practices (39%) were more likely than certified practices to be rural, but their patient populations were similar. Certified practices had more medical home practice systems (79.2% vs 74.9%, P =.01) and were more likely to meet a composite measure of optimal diabetes care (46.8% vs 43.2%, P <.001). A 1-SD increase in presence of practice systems was associated with a 1.4% higher probability of meeting that measure (P <.001). CONCLUSIONS: Practices certified as medical homes have more practice systems and higher performance on diabetes care than uncertified practices, but there is extensive overlap, and any differences may reflect self-selection for certification.

Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs.

The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. Here we analyse bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. Our study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.

Predicting diabetes clinical outcomes using longitudinal risk factor trajectories.

BACKGROUND: The ubiquity of electronic health records (EHR) offers an opportunity to observe trajectories of laboratory results and vital signs over long periods of time. This study assessed the value of risk factor trajectories available in the electronic health record to predict incident type 2 diabetes. STUDY DESIGN AND METHODS: Analysis was based on a large 13-year retrospective cohort of 71,545 adult, non-diabetic patients with baseline in 2005 and median follow-up time of 8 years. The trajectories of fasting plasma glucose, lipids, BMI and blood pressure were computed over three time frames (2000-2001, 2002-2003, 2004) before baseline. A novel method, Cumulative Exposure (CE), was developed and evaluated using Cox proportional hazards regression to assess risk of incident type 2 diabetes. We used the Framingham Diabetes Risk Scoring (FDRS) Model as control. RESULTS: The new model outperformed the FDRS Model (.802 vs .660; p-values <2e-16). Cumulative exposure measured over different periods showed that even short episodes of hyperglycemia increase the risk of developing diabetes. Returning to normoglycemia moderates the risk, but does not fully eliminate it. The longer an individual maintains glycemic control after a hyperglycemic episode, the lower the subsequent risk of diabetes. CONCLUSION: Incorporating risk factor trajectories substantially increases the ability of clinical decision support risk models to predict onset of type 2 diabetes and provides information about how risk changes over time.

Neural-specific Sox2 input and differential Gli-binding affinity provide context and positional information in Shh-directed neural patterning.

In the vertebrate neural tube, regional Sonic hedgehog (Shh) signaling invokes a time- and concentration-dependent induction of six different cell populations mediated through Gli transcriptional regulators. Elsewhere in the embryo, Shh/Gli responses invoke different tissue-appropriate regulatory programs. A genome-scale analysis of DNA binding by Gli1 and Sox2, a pan-neural determinant, identified a set of shared regulatory regions associated with key factors central to cell fate determination and neural tube patterning. Functional analysis in transgenic mice validates core enhancers for each of these factors and demonstrates the dual requirement for Gli1 and Sox2 inputs for neural enhancer activity. Furthermore, through an unbiased determination of Gli-binding site preferences and analysis of binding site variants in the developing mammalian CNS, we demonstrate that differential Gli-binding affinity underlies threshold-level activator responses to Shh input. In summary, our results highlight Sox2 input as a context-specific determinant of the neural-specific Shh response and differential Gli-binding site affinity as an important cis-regulatory property critical for interpreting Shh morphogen action in the mammalian neural tube.

A direct fate exclusion mechanism by Sonic hedgehog-regulated transcriptional repressors.

Sonic hedgehog (Shh) signaling patterns the vertebrate spinal cord by activating a group of transcriptional repressors in distinct neural progenitors of somatic motor neuron and interneuron subtypes. To identify the action of this network, we performed a genome-wide analysis of the regulatory actions of three key ventral determinants in mammalian neural tube patterning: Nkx2.2, Nkx6.1 and Olig2. Previous studies have demonstrated that each factor acts predominantly as a transcriptional repressor, at least in part, to inhibit alternative progenitor fate choices. Here, we reveal broad and direct repression of multiple alternative fates as a general mechanism of repressor action. Additionally, the repressor network targets multiple Shh signaling components providing negative feedback to ongoing Shh signaling. Analysis of chromatin organization around Nkx2.2-, Nkx6.1- and Olig2-bound regions, together with co-analysis of engagement of the transcriptional activator Sox2, indicate that repressors bind to, and probably modulate the action of, neural enhancers. Together, the data suggest a model for neural progenitor specification downstream of Shh signaling, in which Nkx2.2 and Olig2 direct repression of alternative neural progenitor fate determinants, an action augmented by the overlapping activity of Nkx6.1 in each cell type. Integration of repressor and activator inputs, notably activator inputs mediated by Sox2, is probably a key mechanism in achieving cell type-specific transcriptional outcomes in mammalian neural progenitor fate specification.

CRISPRtools: a flexible computational platform for performing CRISPR/Cas9 experiments in the mouse.

Genome editing using the CRISPR/Cas9 RNA-guided endonuclease system has rapidly become a driving force for discovery in modern biomedical research. This simple yet elegant system has been widely used to generate both loss-of-function alleles and precision knock-in mutations using single-stranded donor oligonucleotides. Our CRISPRtools platform supports both of these applications in order to facilitate the use of CRISPR/Cas9. While there are several tools that facilitate CRISPR/Cas9 design and screen for potential off-target sites, the process is typically performed sequentially on single genes, limiting scalability for large-scale programs. Here, the design principle underlying gene ablation is based upon using paired guides flanking a critical region/exon of interest to create deletions. Guide pairs are rank ordered based upon published efficiency scores and off-target analyses, and reported in a concise format for downstream implementation. The exon deletion strategy simplifies characterization of founder animals and is the strategy employed for the majority of knockouts in the mouse. In proof-of-principle experiments, the effectiveness of this approach is demonstrated using microinjection and electroporation to introduce CRISPR/Cas9 components into mouse zygotes to delete critical exons.

Essential role for ligand-dependent feedback antagonism of vertebrate hedgehog signaling by PTCH1, PTCH2 and HHIP1 during neural patterning.

Hedgehog (HH) signaling is essential for vertebrate and invertebrate embryogenesis. In Drosophila, feedback upregulation of the HH receptor Patched (PTC; PTCH in vertebrates), is required to restrict HH signaling during development. By contrast, PTCH1 upregulation is dispensable for early HH-dependent patterning in mice. Unique to vertebrates are two additional HH-binding antagonists that are induced by HH signaling, HHIP1 and the PTCH1 homologue PTCH2. Although HHIP1 functions semi-redundantly with PTCH1 to restrict HH signaling in the developing nervous system, a role for PTCH2 remains unresolved. Data presented here define a novel role for PTCH2 as a ciliary localized HH pathway antagonist. While PTCH2 is dispensable for normal ventral neural patterning, combined removal of PTCH2- and PTCH1-feedback antagonism produces a significant expansion of HH-dependent ventral neural progenitors. Strikingly, complete loss of PTCH2-, HHIP1- and PTCH1-feedback inhibition results in ectopic specification of ventral cell fates throughout the neural tube, reflecting constitutive HH pathway activation. Overall, these data reveal an essential role for ligand-dependent feedback inhibition of vertebrate HH signaling governed collectively by PTCH1, PTCH2 and HHIP1.

A practical Bayesian stepped wedge design for community-based cluster-randomized clinical trials: The British Columbia Telehealth Trial.

BACKGROUND: Many clinical trial designs are impractical for community-based clinical intervention trials. Stepped wedge trial designs provide practical advantages, but few descriptions exist of their clinical implementational features, statistical design efficiencies, and limitations. OBJECTIVES: Enhance efficiency of stepped wedge trial designs by evaluating the impact of design characteristics on statistical power for the British Columbia Telehealth Trial. METHODS: The British Columbia Telehealth Trial is a community-based, cluster-randomized, controlled clinical trial in rural and urban British Columbia. To determine the effect of an Internet-based telehealth intervention on healthcare utilization, 1000 subjects with an existing diagnosis of congestive heart failure or type 2 diabetes will be enrolled from 50 clinical practices. Hospital utilization is measured using a composite of disease-specific hospital admissions and emergency visits. The intervention comprises online telehealth data collection and counseling provided to support a disease-specific action plan developed by the primary care provider. The planned intervention is sequentially introduced across all participating practices. We adopt a fully Bayesian, Markov chain Monte Carlo-driven statistical approach, wherein we use simulation to determine the effect of cluster size, sample size, and crossover interval choice on type I error and power to evaluate differences in hospital utilization. RESULTS: For our Bayesian stepped wedge trial design, simulations suggest moderate decreases in power when crossover intervals from control to intervention are reduced from every 3 to 2 weeks, and dramatic decreases in power as the numbers of clusters decrease. Power and type I error performance were not notably affected by the addition of nonzero cluster effects or a temporal trend in hospitalization intensity. CONCLUSION/LIMITATIONS: Stepped wedge trial designs that intervene in small clusters across longer periods can provide enhanced power to evaluate comparative effectiveness, while offering practical implementation advantages in geographic stratification, temporal change, use of existing data, and resource distribution. Current population estimates were used; however, models may not reflect actual event rates during the trial. In addition, temporal or spatial heterogeneity can bias treatment effect estimates.

Gene regulatory networks mediating canonical Wnt signal-directed control of pluripotency and differentiation in embryo stem cells.

Canonical Wnt signaling supports the pluripotency of embryonic stem cells (ESCs) but also promotes differentiation of early mammalian cell lineages. To explain these paradoxical observations, we explored the gene regulatory networks at play. Canonical Wnt signaling is intertwined with the pluripotency network comprising Nanog, Oct4, and Sox2 in mouse ESCs. In defined media supporting the derivation and propagation of ESCs, Tcf3 and ß-catenin interact with Oct4; Tcf3 binds to Sox motif within Oct-Sox composite motifs that are also bound by Oct4-Sox2 complexes. Furthermore, canonical Wnt signaling upregulates the activity of the Pou5f1 distal enhancer via the Sox motif in ESCs. When viewed in the context of published studies on Tcf3 and ß-catenin mutants, our findings suggest Tcf3 counters pluripotency by competition with Sox2 at these sites, and Tcf3 inhibition is blocked by ß-catenin entry into this complex. Wnt pathway stimulation also triggers ß-catenin association at regulatory elements with classic Lef/Tcf motifs associated with differentiation programs. The failure to activate these targets in the presence of a mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor essential for ESC culture suggests MEK/ERK signaling and canonical Wnt signaling combine to promote ESC differentiation.

A 16-month community-based intervention to increase aspirin use for primary prevention of cardiovascular disease.

INTRODUCTION: Cardiovascular diseases are the leading causes of disability and death in the United States. Primary prevention of these events may be achieved through aspirin use. The ability of a community-based intervention to increase aspirin use has not been evaluated. The objective of this study was to evaluate an educational intervention implemented to increase aspirin use for primary prevention of cardiovascular disease in a small city in Minnesota. METHODS: A community-based intervention was implemented during 16 months in a medium-sized community in Minnesota. Messages for aspirin use were disseminated to individuals, health care professionals, and the general population. Independent cross-sectional samples of residents (men aged 45-79, women aged 55-79) were surveyed by telephone to identify candidates for primary prevention aspirin use, examine their characteristics, and determine regular aspirin use at baseline and after the campaign at 4 months and 16 months. RESULTS: In primary prevention candidates, regular aspirin use rates increased from 36% at baseline to 54% at 4 months (odds ratio = 2.05; 95% confidence interval, 1.09-3.88); the increase was sustained at 52% at 16 months (odds ratio = 1.89; 95% confidence interval, 1.02-3.49). The difference in aspirin use rates at 4 months and 16 months was not significant (P = .77). CONCLUSION: Aspirin use rates for primary prevention remain low. A combined public health and primary care approach can increase and sustain primary prevention aspirin use in a community setting.