Building on existing tools to improve chronic disease prevention and screening in public health: a cluster randomized trial.

BACKGROUND: The BETTER (Building on Existing Tools to Improve Chronic Disease Prevention and Screening in Primary Care) intervention was designed to integrate the approach to chronic disease prevention and screening in primary care and demonstrated effective in a previous randomized trial. METHODS: We tested the effectiveness of the BETTER HEALTH intervention, a public health adaptation of BETTER, at improving participation in chronic disease prevention and screening actions for residents of low-income neighbourhoods in a cluster randomized trial, with ten low-income neighbourhoods in Durham Region Ontario randomized to immediate intervention vs. wait-list. The unit of analysis was the individual, and eligible participants were adults age 40-64 years residing in the neighbourhoods. Public health nurses trained as "prevention practitioners" held one prevention-focused visit with each participant. They provided participants with a tailored prevention prescription and supported them to set health-related goals. The primary outcome was a composite index: the number of evidence-based actions achieved at six months as a proportion of those for which participants were eligible at baseline. RESULTS: Of 126 participants (60 in immediate arm; 66 in wait-list arm), 125 were included in analyses (1 participant withdrew consent). In both arms, participants were eligible for a mean of 8.6 actions at baseline. At follow-up, participants in the immediate intervention arm met 64.5% of actions for which they were eligible versus 42.1% in the wait-list arm (rate ratio 1.53 [95% confidence interval 1.22-1.84]). CONCLUSION: Public health nurses using the BETTER HEALTH intervention led to a higher proportion of identified evidence-based prevention and screening actions achieved at six months for people living with socioeconomic disadvantage. TRIAL REGISTRATION: NCT03052959 , registered February 10, 2017.

What is a prevention visit? A qualitative study of a structured approach to prevention and screening - the BETTER WISE project.

BACKGROUND: This qualitative study is a sub-component of BETTER WISE, a comprehensive and structured approach that proactively addresses chronic disease prevention, screening, and cancer survivorship, including screening for poverty and addressing lifestyle risks for patients aged 40 to 65. Patients (n = 527) from 13 primary care clinics (urban, rural, and remote) in Alberta, Ontario, and Newfoundland & Labrador, Canada agreed to participate in the study and were invited to a one-hour prevention visit delivered by a Prevention Practitioner (PP) as part of BETTER WISE. We identified the key components of a BETTER WISE prevention visit based on patients' and primary care providers' perspectives. METHODS: Primary care providers (PPs, physicians and their staff) participated in 14 focus groups and 19 key informant interviews to share their perspectives on the BETTER WISE project. Of 527 patients who agreed to participate in the study and were invited for a BETTER WISE prevention visit with a PP, we received 356 patient feedback forms. We also collected field notes and memos and employed thematic analysis using a constant comparative method focusing on the BETTER WISE prevention visit. RESULTS: We identified four key themes related to a BETTER WISE prevention visit: 1) Creating a safe environment and building trust with patients: PPs provided sufficient time and a safe space for patients to share what was important to them, including their concerns related to poverty, alcohol consumption, and mental health, topics that were often not shared with physicians; 2) Providing personalized health education: PPs used the BETTER WISE tools to provide patients with a personalized overview of their health status and eligible screening; 3) Non-judgmental empowering of patients: Instead of directing patients on what to do, PPs evoked patients' preferences and helped them to set goals (if desired); and 4) Integrating care for patients: PPs clarified information from patients' charts and surveys with physicians and helped patients to navigate resources within and outside of the primary care team. CONCLUSIONS: The results of this study underscore the importance of personalized, trusting, non-judgmental, and integrated relationships between primary care providers and patients to effectively address chronic disease prevention, screening, and cancer survivorship as demonstrated by the BETTER WISE prevention visits. TRIAL REGISTRATION: This qualitative study is a sub-component of the BETTER WISE pragmatic, cRCT, trial registration ISRCTN21333761 (date of registration 19/12/2016).

The role of family physicians in cancer care: perspectives of primary and specialty care providers.

BACKGROUND: Currently, the specific role of family physicians (fps) in the care of people with cancer is not well defined. Our goal was to explore physician perspectives and contextual factors related to the coordination of cancer care and the role of fps. METHODS: Using a constructivist grounded theory approach, we conducted telephone interviews with 58 primary and cancer specialist health care providers from across Canada. RESULTS: The participants-21 fps, 15 surgeons, 12 medical oncologists, 6 radiation oncologists, and 4 general practitioners in oncology-were asked to describe both the role that fps currently play and the role that, in their opinion, fps should play in the future care of cancer patients across the cancer continuum. Participants identified 3 key roles: coordinating cancer care, managing comorbidities, and providing psychosocial care to patients and their families. However, fps and specialists discussed many challenges that prevent fps from fully performing those roles: ■ The fps described communication problems resulting from not being kept "in the loop" because they weren't copied on patient reports and also the lack of clearly defined roles for all the various health care providers involved in providing care to cancer patients.■ The specialists expressed concerns about a lack of patient access to fp care, leaving specialists to fill the care gaps. The fps and specialists both recommended additional training and education for fps in survivorship care, cancer screening, genetic testing, and new cancer treatments. CONCLUSIONS: Better communication, more collaboration, and further education are needed to enhance the role of fps in the care of cancer patients.

Uptake of a 21-gene expression assay in breast cancer practice: views of academic and community-based oncologists.

PURPOSE: Advances in personalized medicine have produced novel tests and treatment options for women with breast cancer. Relatively little is known about the process by which such tests are adopted into oncology practice. The objectives of the present study were to understand the experiences of medical oncologists with multigene expression profile (gep) tests, including their adoption into practice in early-stage breast cancer, and the perceptions of the oncologists about the influence of test results on treatment decision-making. METHODS: We conducted a qualitative descriptive study involving interviews with medical oncologists from academic and community cancer centres or hospitals in 8 communities in Ontario. A 21-gene breast cancer assay was used as the example of gep testing. Qualitative analytic techniques were used to identify the main themes. RESULTS: Of 28 oncologists who were approached, 21 (75%) participated in the study [median age: 43 years; 12 women (57%)]. Awareness and knowledge of gep testing were derived from several sources: international scientific meetings, participation in clinical studies, discussions with respected colleagues, and manufacturer-sponsored meetings. Oncologists observed that incorporating gep testing into their clinical practice resulted in several changes, including longer consultation times, second visits, and taking steps to minimize treatment delays. Oncologists expressed divergent opinions about the strength of evidence and added value of gep testing in guiding treatment decisions. CONCLUSIONS: Incorporation of gep testing into clinical practice in early-stage breast cancer required oncologists to make changes to their usual routines. The opinions of oncologists about the quality of evidence underpinning the test affected how much weight they gave to test results in treatment decision-making.

Challenges and insights in implementing coordinated care between oncology and primary care providers: a Canadian perspective.

We report here on the current state of cancer care coordination in Canada and discuss challenges and insights with respect to the implementation of collaborative models of care. We also make recommendations for future research. This work is based on the findings of the Canadian Team to Improve Community-Based Cancer Care Along the Continuum (canimpact) casebook project. The casebook project identified models of collaborative cancer care by systematically documenting and analyzing Canadian initiatives that aim to improve or enhance care coordination between primary care providers and oncology specialists. The casebook profiles 24 initiatives, most of which focus on breast or colorectal cancer and target survivorship or follow-up care. Current key challenges in cancer care coordination are associated with establishing program support, engaging primary care providers in the provision of care, clearly defining provider roles and responsibilities, and establishing effective project or program planning and evaluation. Researchers studying coordinated models of cancer care should focus on designing knowledge translation strategies with updated and refined governance and on establishing appropriate protocols for both implementation and evaluation.

Multigene expression profile testing in breast cancer: is there a role for family physicians?

BACKGROUND: Family physicians (fps) play a role in aspects of personalized medicine in cancer, including assessment of increased risk because of family history. Little is known about the potential role of fps in supporting cancer patients who undergo tumour gene expression profile (gep) testing. METHODS: We conducted a mixed-methods study with qualitative and quantitative components. Qualitative data from focus groups and interviews with fps and cancer specialists about the role of fps in breast cancer gep testing were obtained during studies conducted within the pan-Canadian canimpact research program. We determined the number of visits by breast cancer patients to a fp between the first medical oncology visit and the start of chemotherapy, a period when patients might be considering results of gep testing. RESULTS: The fps and cancer specialists felt that ordering gep tests and explaining the results was the role of the oncologist. A new fp role was identified relating to the fp-patient relationship: supporting patients in making adjuvant therapy decisions informed by gep tests by considering the patient's comorbid conditions, social situation, and preferences. Lack of fp knowledge and resources, and challenges in fp-oncologist communication were seen as significant barriers to that role. Between 28% and 38% of patients visited a fp between the first oncology visit and the start of chemotherapy. CONCLUSIONS: Our findings suggest an emerging role for fps in supporting patients who are making adjuvant treatment decisions after receiving the results of gep testing. For success in this new role, education and point-of-care tools, together with more effective communication strategies between fps and oncologists, are needed.

Views of family physicians about survivorship care plans to provide breast cancer follow-up care: exploration of results from a randomized controlled trial.

BACKGROUND: The U.S. Institute of Medicine recommends that cancer patients receive survivorship care plans, but evaluations to date have found little evidence of the effectiveness of such plans. We conducted a qualitative follow-on study to a randomized controlled trial (rct) to understand the experiences of family physicians using survivorship care plans to support the follow-up of breast cancer patients. METHODS: A subset of family physicians whose patients were enrolled in the parent rct in Ontario and Nova Scotia were eligible for this study. In interviews, the physicians discussed survivorship care plans (intervention) or usual discharge letters (control), and their confidence in providing follow-up cancer care. RESULTS: Of 123 eligible family physicians, 18 (10 intervention, 8 control) were interviewed. In general, physicians receiving a survivorship care plan found only the 1-page care record to be useful. Physicians who received only a discharge letter had variable views about the letter's usefulness; several indicated that it lacked information about potential cancer- or treatment-related problems. Most physicians were comfortable providing care 3-5 years after diagnosis, but desired timely and informative communication with oncologists. CONCLUSIONS: Although family physicians did not find extensive survivorship care plans useful, discharge letters might not be sufficiently comprehensive for follow-up breast cancer care. Effective strategies for two-way communication between family physicians and oncologists are still lacking.

Lessons learned from a cancer knowledge translation grants program: results of an evaluation.

Background: A novel way to build capacity in knowledge translation (kt) is through kt-focused grant competitions. Since 2009, the Knowledge Translation Research Network (KT-Net) has had a cancer-related kt grants program. We undertook an evaluation of the program to determine if KT-Net was achieving its aims of building capacity in cancer kt, advancing the science of kt, building partnerships, and leveraging funding. Methods: An adapted framework guided the evaluation. Nine funded studies from 4 competitions were included. Semi-structured telephone interviews were held with researchers, stakeholders (including knowledge users), members of grant review panels, and experts in kt. Interview transcripts were audio-recorded, transcribed, and analyzed thematically. A review of proposal and report documents was also conducted. Results: Funded researchers indicated that the grant competition was an essential funding program for cancer kt research. Competitions were perceived to build capacity in cancer kt among early-career researchers and to encourage innovative cancer kt research for which alternative funding sources are limited. The grants program resulted in incremental gains in advancing the science of kt. Suggestions to improve the program included stronger partnerships between the funder and the provincial cancer-system organization to optimize the application of research that is relevant to the organization's strategic objectives. Conclusions: The grants program met many of its aims by providing cancer researchers with an opportunity to gain capacity in cancer kt and by making incremental advances in kt science. Suggestions to improve the program included closer partnerships between the funder and the cancer-system organization.

Cost-effectiveness of novel diagnostic tools for the diagnosis of tuberculosis.

SETTING: The potential cost-effectiveness of improved diagnostic tests for tuberculosis (TB) in resource-limited settings is unknown. OBJECTIVE: To estimate the incremental cost-effectiveness of a hypothetical new point-of-care TB diagnostic test in South Africa, Brazil and Kenya. DESIGN: Decision-analysis model, adding four diagnostic interventions (sputum smear microscopy, new test, smear plus new test and smear plus TB culture) to a baseline of existing infrastructure without smear. RESULTS: Adding sputum smear was estimated to be more cost-effective (incremental cost per disability-adjusted life year [DALY] of $86 [South Africa], $131 [Brazil], $38 (Kenya]) than a new TB diagnostic with 70% sensitivity, 95% specificity and price of $20 per test ($198 [South Africa], $275 [Brazil], $84 [Kenya]). However, compared to sputum smear, smear plus new test averted 46-49% more DALYs per 1000 TB suspects (321 vs. 215 [South Africa], 243 vs. 166 [Brazil], 790 vs. 531 [Kenya]), at an incremental cost of $170 (Kenya) to $625 (Brazil) per DALY averted. Cost-effectiveness was most sensitive to the specificity and price of the new test, the baseline TB case detection rate and the discount rate. CONCLUSION: Novel diagnostic tests for TB are potentially highly cost-effective. Cost-effectiveness is maximized by high-specificity, low-cost tests deployed to regions with poor infrastructure.

25-Hydroxyvitamin D concentrations in dogs with naturally acquired blastomycosis.

BACKGROUND: Hypovitaminosis D is common in humans with tuberculosis, and adequate serum 25-hydroxyvitamin D [25(OH)D] concentrations may improve response to therapy. The pathomechanism of Blastomyces dermatitidis is similar to that of Mycobacterium tuberculosis, but the 25(OH)D status of dogs with blastomycosis has not been investigated. OBJECTIVES: To determine if dogs with blastomycosis have lower 25(OH)D concentrations compared with healthy controls and to explore the prognostic value of 25(OH)D concentrations in blastomycosis. ANIMALS: 35 control dogs (16 client-owned, healthy dogs and 19 healthy, random-source hound mixes) and 22 dogs with blastomycosis. METHODS: Prospective study. Serum concentrations of 25(OH)D, parathyroid hormone (PTH), ionized calcium were measured, and biochemistry and hematology profiles were performed. The 25-hydroxyvitamin D concentrations were compared between groups, and factors associated with 25(OH)D variation were investigated in dogs with blastomycosis. Dogs with blastomycosis were followed for up to 5 years after discharge and factors associated with survival were investigated. RESULTS: Dogs with blastomycosis had significantly lower concentrations of 25(OH)D and PTH and higher concentrations of ionized calcium than did control dogs. In dogs with blastomycosis, 25(OH)D concentrations were independently associated with neutrophil count, pCO2 , and with bone and skin involvement. The 25-hydroxyvitamin D concentration was not associated with survival in dogs with blastomycosis, whereas lactate concentrations; bone, skin, and lymph node involvement; number of affected sites; and, presence of respiratory signs were associated with survival. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with blastomycosis had lower 25(OH)D concentrations than did healthy controls. Despite no impact on survival, investigating the effect of 25(OH)D supplementation on recovery is warranted.

Local opinion leaders: effects on professional practice and health care outcomes.

BACKGROUND: Clinical practice is not always evidence-based and, therefore, may not optimise patient outcomes. Opinion leaders disseminating and implementing 'best evidence' is one innovative method that holds promise as a strategy to bridge evidence-practice gaps. OBJECTIVES: To assess the effectiveness of the use of local opinion leaders in improving the behaviour of health care professionals and patient outcomes. SEARCH STRATEGY: We searched MEDLINE, Health Star, SIGLE and the Cochrane Effective Practice and Organisation of Care Group Trials Register. We did not apply date restrictions to our search strategy. Searches were last updated in February 2005. In addition, we searched reference lists of all potential studies that were identified. SELECTION CRITERIA: Studies eligible for inclusion were randomized controlled trials that used objective measures of performance/provider behaviour and/or patient health outcomes. DATA COLLECTION AND ANALYSIS: Two reviewers extracted data from each study and assessed its methodological quality. We calculated the absolute difference in the risk of 'non-compliance' with desired practice, adjusting for baseline levels of non-compliance where these data were available. MAIN RESULTS: Twelve studies met our eligibility criteria. The adjusted absolute risk difference of non-compliance with desired practice varied from -6% (favouring control) to +25% (favouring opinion leader intervention). Overall, the median adjusted risk difference (ARD) was 0.10 representing a 10% absolute decrease in non-compliance in the intervention group. AUTHORS' CONCLUSIONS: The use of local opinion leaders can successfully promote evidence-based practice. However the feasibility of its widespread use remains uncertain.

WITHDRAWN: Audit and feedback versus alternative strategies: effects on professional practice and health care outcomes.

BACKGROUND: Audit and feedback has been identified as having the potential to change the practice of health care professionals. OBJECTIVES: To assess the effects of audit and feedback compared with other interventions in changing health professional practice and to assess whether the effectiveness of audit and feedback can be improved by modifying how it is done. SEARCH STRATEGY: We searched MEDLINE up to June 1997, the Research and Development Resource Base in Continuing Medical Education, and reference lists of related systematic reviews and articles. SELECTION CRITERIA: Randomised trials of audit and feedback (defined as any summary of clinical performance of health care over a specified period of time) compared with other interventions. The participants were health care providers responsible for patient care. DATA COLLECTION AND ANALYSIS: Two reviewers independently extracted data and assessed study quality. MAIN RESULTS: Twelve studies were included involving more than 2194 physicians. Seven trials with direct comparisons were included. The targeted behaviours were the management of low haemoglobin, the delivery of preventive care services (two studies), the management of high cholesterol, the performance of cervical smears, and the ordering of diagnostic tests (two studies). From the results of four trials, there is little evidence of a measurable effect of adding a complementary intervention such as a local consensus process to audit and feedback compared to audit and feedback alone. Two of three trials that compared audit and feedback to reminders reported that reminders were more effective in improving the delivery of some preventive services. AUTHORS' CONCLUSIONS: It is not possible to recommend a complementary intervention to enhance the effectiveness of audit and feedback. Reminders might be more effective than audit and feedback to improve the delivery of some preventive services but the results are not striking. Few trials have investigated the effect of varying different characteristics of the audit and feedback process. Consideration should be given to testing the effects of modifying important characteristics such as the content, source, timing, recipient and format.

Educational outreach visits: effects on professional practice and health care outcomes.

BACKGROUND: Educational outreach visits (EOVs) have been identified as an intervention that may improve the practice of healthcare professionals. This type of face-to-face visit has been referred to as university-based educational detailing, academic detailing, and educational visiting. OBJECTIVES: To assess the effects of EOVs on health professional practice or patient outcomes. SEARCH STRATEGY: For this update, we searched the Cochrane EPOC register to March 2007. In the original review, we searched multiple bibliographic databases including MEDLINE and CINAHL. SELECTION CRITERIA: Randomised trials of EOVs that reported an objective measure of professional performance or healthcare outcomes. An EOV was defined as a personal visit by a trained person to healthcare professionals in their own settings. DATA COLLECTION AND ANALYSIS: Two reviewers independently extracted data and assessed study quality. We used bubble plots and box plots to visually inspect the data. We conducted both quantitative and qualitative analyses. We used meta-regression to examine potential sources of heterogeneity determined a priori. We hypothesised eight factors to explain variation across effect estimates. In our primary visual and statistical analyses, we included only studies with dichotomous outcomes, with baseline data and with low or moderate risk of bias, in which the intervention included an EOV and was compared to no intervention. MAIN RESULTS: We included 69 studies involving more than 15,000 health professionals. Twenty-eight studies (34 comparisons) contributed to the calculation of the median and interquartile range for the main comparison. The median adjusted risk difference (RD) in compliance with desired practice was 5.6% (interquartile range 3.0% to 9.0%). The adjusted RDs were highly consistent for prescribing (median 4.8%, interquartile range 3.0% to 6.5% for 17 comparisons), but varied for other types of professional performance (median 6.0%, interquartile range 3.6% to 16.0% for 17 comparisons). Meta-regression was limited by the large number of potential explanatory factors (eight) with only 31 comparisons, and did not provide any compelling explanations for the observed variation in adjusted RDs. There were 18 comparisons with continuous outcomes, with a median adjusted relative improvement of 21% (interquartile range 11% to 41%). There were eight trials (12 comparisons) in which the intervention included an EOV and was compared to another type of intervention, usually audit and feedback. Interventions that included EOVs appeared to be slightly superior to audit and feedback. Only six studies evaluated different types of visits in head-to-head comparisons. When individual visits were compared to group visits (three trials), the results were mixed. AUTHORS' CONCLUSIONS: EOVs alone or when combined with other interventions have effects on prescribing that are relatively consistent and small, but potentially important. Their effects on other types of professional performance vary from small to modest improvements, and it is not possible from this review to explain that variation.

Audit and feedback: effects on professional practice and health care outcomes.

BACKGROUND: Audit and feedback continues to be widely used as a strategy to improve professional practice. It appears logical that healthcare professionals would be prompted to modify their practice if given feedback that their clinical practice was inconsistent with that of their peers or accepted guidelines. Yet, audit and feedback has not consistently been found to be effective. OBJECTIVES: To assess the effects of audit and feedback on the practice of healthcare professionals and patient outcomes. SEARCH STRATEGY: We searched the Cochrane Effective Practice and Organisation of Care Group's register and pending file up to January 2004. SELECTION CRITERIA: Randomised trials of audit and feedback (defined as any summary of clinical performance over a specified period of time) that reported objectively measured professional practice in a healthcare setting or healthcare outcomes. DATA COLLECTION AND ANALYSIS: Two reviewers independently extracted data and assessed study quality. Quantitative (meta-regression), visual and qualitative analyses were undertaken. For each comparison we calculated the risk difference (RD) and risk ratio (RR), adjusted for baseline compliance when possible, for dichotomous outcomes and the percentage and the percent change relative to the control group average after the intervention, adjusted for baseline performance when possible, for continuous outcomes. We investigated the following factors as possible explanations for the variation in the effectiveness of interventions across comparisons: the type of intervention (audit and feedback alone, audit and feedback with educational meetings, or multifaceted interventions that included audit and feedback), the intensity of the audit and feedback, the complexity of the targeted behaviour, the seriousness of the outcome, baseline compliance and study quality. MAIN RESULTS: Thirty new studies were added to this update, and a total of 118 studies are included. In the primary analysis 88 comparisons from 72 studies were included that compared any intervention in which audit and feedback is a component compared to no intervention. For dichotomous outcomes the adjusted risk difference of compliance with desired practice varied from - 0.16 (a 16 % absolute decrease in compliance) to 0.70 (a 70% increase in compliance) (median = 0.05, inter-quartile range = 0.03 to 0.11) and the adjusted risk ratio varied from 0.71 to 18.3 (median = 1.08, inter-quartile range = 0.99 to 1.30). For continuous outcomes the adjusted percent change relative to control varied from -0.10 (a 10 % absolute decrease in compliance) to 0.68 (a 68% increase in compliance) (median = 0.16, inter-quartile range = 0.05 to 0.37). Low baseline compliance with recommended practice and higher intensity of audit and feedback were associated with larger adjusted risk ratios (greater effectiveness) across studies. AUTHORS' CONCLUSIONS: Audit and feedback can be effective in improving professional practice. When it is effective, the effects are generally small to moderate. The relative effectiveness of audit and feedback is likely to be greater when baseline adherence to recommended practice is low and when feedback is delivered more intensively.

Multiple amidated neuropeptides are required for normal circadian locomotor rhythms in Drosophila.

In Drosophila, the amidated neuropeptide pigment dispersing factor (PDF) is expressed by the ventral subset of lateral pacemaker neurons and is required for circadian locomotor rhythms. Residual rhythmicity in pdf mutants likely reflects the activity of other neurotransmitters. We asked whether other neuropeptides contribute to such auxiliary mechanisms. We used the gal4/UAS system to create mosaics for the neuropeptide amidating enzyme PHM; amidation is a highly specific and widespread modification of secretory peptides in Drosophila. Three different gal4 drivers restricted PHM expression to different numbers of peptidergic neurons. These mosaics displayed aberrant locomotor rhythms to degrees that paralleled the apparent complexity of the spatial patterns. Certain PHM mosaics were less rhythmic than pdf mutants and as severe as per mutants. Additional gal4 elements were added to the weakly rhythmic PHM mosaics. Although adding pdf-gal4 provided only partial improvement, adding the widely expressed tim-gal4 largely restored rhythmicity. These results indicate that, in Drosophila, peptide amidation is required for neuropeptide regulation of behavior. They also support the hypothesis that multiple amidated neuropeptides, acting upstream, downstream, or in parallel to PDF, help organize daily locomotor rhythms.

A genetic and molecular analysis of the 46C chromosomal region surrounding the FMRFamide neuropeptide gene in Drosophila melanogaster.

We have analyzed the FMRFamide neuropeptide gene region of Drosophila melanogaster. This gene maps to the 46C region of chromosome 2R; this interval previously was not well characterized. For this genetic and molecular analysis, we have used X-ray mutagenesis, EMS mutagenesis, and the recently reported local P element transposition method. We identified four overlapping deletions, two of which have proximal breakpoints that define a 50-60-kb region surrounding the FMRFamide gene in 46C. To this small region, we mapped three lethal complementation groups; 10 additional lethal complementation groups were mapped to more distal regions of 46CD. One of these groups corresponds to even-skipped, the other 12 are previously unidentified. Using various lines of evidence we excluded the possibility that FMRFamide corresponds to any of the three lethal complementation groups mapping to its immediate 50-60-kb vicinity. The positions of two of the three lethal complementation groups were identified with P elements using a local transposition scheme. The third lethal complementation group was excluded as being FMRFamide mutants by sequence analysis and by immunocytochemistry with proFMRFamide precursor-specific antibodies. This analysis has (1) provided a genetic map of the 46CD chromosomal region and a detailed molecular map of a portion of the 46C region and (2) provided additional evidence of the utility of local transposition for targeting nearby genes.

In situ hybridization analysis of the FMRFamide neuropeptide gene in Drosophila. I. Restricted expression in embryonic and larval stages.

We have used in situ hybridization techniques to describe the cellular distribution of transcripts from a Drosophila gene that encodes multiple FMRFamide-related neuropeptides. The Drosophila FMRFamide gene consists of two exons and is expressed predominantly as a approximately 1.7 kb RNA throughout postembryonic stages (Nambu et al., '88; Schneider and Taghert, '88, '90). We used exon-specific oligonucleotide probes to assay transcription in both embryonic and larval stages and found a pattern of hybridization signals that was restricted to the central nervous system and, within that tissue, was cell-specific. The pattern included 36 distinct signals distributed throughout both the brain and segmental nerve cord (ventral ganglion). These observations suggest that the cell-specific pattern of FMRFamide-like neuropeptide expression in the Drosophila CNS (White et al., '86; Taghert and Schneider, '90) is due to the restricted expression of specific gene transcripts. The results also indicate that, with few exceptions, all previously identified FMRFamide-immunoreactive neurons in Drosophila larvae express FMRFamide gene transcripts. The 36 hybridization regions of the CNS could be divided into three categories, based on their signal intensities (strong, moderate, and weak). The differences in intensity were reproducible and suggest that steady-state levels of specific neuropeptide RNA differ among individual neurons. The two exon-specific probes produced patterns that were indistinguishable both in pattern and in intensity. This result supports the previous conclusion that the one detectable FMRFamide transcript contains both exons (Schneider and Taghert, '90). A single identifiable signal was detected during embryogenesis (beginning at stage 16), but the mature complement of signals was not fully established until the final larval stages.

In situ hybridization analysis of the FMRFamide neuropeptide gene in Drosophila. II. Constancy in the cellular pattern of expression during metamorphosis.

We have studied changes in the pattern of specific neuropeptide gene expression during the metamorphosis of the Drosophila nervous system. Prior to metamorphosis, the Drosophila FMRFamide gene is expressed exclusively within the central nervous system in a stereotyped pattern that comprises roughly 60 neurons (Schneider et al., '91). Using in situ hybridization, we found that the FMRFamide gene was continuously expressed throughout all stages examined: at each of 15 stages of adult development and through at least the first 10 days of adult life. There were no differences between the results observed with 2 exon-specific hybridization probes, thus indicating little if any alternative splicing during postembryonic development. Despite many changes in the positions of individual hybridization signals due to the large-scale reorganization of the nervous system, the continuous pattern of gene expression through adult development permitted many adult signals to be identified as larval signals. We concluded that the adult pattern of FMRFamide gene expression was largely derived from persistent larval neurons. Adult-specific hybridization signals in the brain and ventral ganglion were also detected and these corresponded to many of the approximately 40 adult-specific FMRFamide-immunoreactive neurons. One specific larval signal was lost during adult development and the intensities of other signals fluctuated in reproducible manners. These stereotyped differences in hybridization signal intensity resemble similar observations made in larval stages (Schneider et al., '91) and support the hypothesis that the steady-state levels of FMRFamide transcripts are differentially regulated among the diverse neurons that express the gene.