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In Nepal, abortion was legalized in 2002. Yet many women are denied abortion services. Women denied abortion services may either continue their pregnancies or find abortion care elsewhere. However, what is not known is the consequences on women, and their children after accessing abortion services or after being denied abortion services. This comment aims to understand the cause of death of women who sought abortion services between 2019 and 2020 and were enrolled in a longitudinal nationwide study of the consequences of legal abortion access in Nepal. Women were interviewed 6 weeks and every 6 months for 3 years after seeking abortion. During the follow-up interviews, the field research assistants were informed about the death of the clients. Once the death was reported, a trained senior research staff visited the deceased persons house and interviewed family members including husbands, maternal parents or in-laws to explore the cause of death. A total of nine deaths were reported between April 2019 and December 2022. Out of nine deceased women, four received abortions while five of them were initially denial abortion services. The majority of the deaths were due to suicide followed by tuberculosis. None of the deaths were caused by abortion or birth. Keywords: Death; Nepal; reproductive ages; womens health.

This article develops the concept of "menstrual justice". The legal scholar Margaret E. Johnson has developed an expansive approach to menstrual justice incorporating rights, justice, and a framework for intersectional analysis, with a focus on the US. This framework provides a welcome alternative to the constrictive and medicalised approaches often taken towards menstruation. However, the framework is silent on several issues pertaining to menstruation in Global South contexts. This article therefore develops the concept of menstrual justice in order to extend its relevance beyond the Global North. It presents the findings of mixed-methods research conducted in April 2019 in the mid-western region of Nepal, particularly concerning the practice of chhaupadi , an extreme form of menstrual restriction. We conducted a quantitative survey of 400 adolescent girls and eight focus group discussions, four with adolescent girls and four with adult women. Our findings confirm that dignity in menstruation requires addressing pain management, security issues, and mental health, plus structural issues including economic disadvantage, environmental issues, criminal law, and education.

This paper examines factors associated with intimate partner violence (IPV) among newly married women in Nepal, and how IPV was affected by food insecurity and COVID-19. Given evidence that food insecurity is associated with IPV and COVID-19, we explored whether increased food insecurity during COVID-19 is associated with changes in IPV. We used data from a cohort study of 200 newly married women aged 18-25 years, interviewed five times over two years at 6-month intervals (02/2018-07/2020), including after COVID-19-associated lockdowns. Bivariate analysis and mixed-effects logistic regression models were used to examine the association between selected risk factors and recent IPV. IPV increased from 24.5% at baseline to 49.2% before COVID-19 and to 80.4% after COVID-19. After adjusting for covariates, we find that both COVID-19 (OR = 2.93, 95% CI 1.07-8.02) and food insecurity (OR = 7.12, 95% CI 4.04-12.56) are associated with increased odds of IPV, and IPV increased more for food-insecure women post COVID-19 (compared to non-food insecure), but this was not statistically significant (confidence interval 0.76-8.69, p -value = 0.131). Young, newly married women experience high rates of IPV that increase with time in marriage, and COVID-19 has exacerbated this, especially for food-insecure women in the present sample. Along with enforcement of laws against IPV, our results suggest that special attention needs to be paid to women during a crisis time like the current COVID-19 pandemic, especially those who experience other household stressors.

Background: Mindful of social norms shaping health among women pressured to prove early fertility in Nepal, a bi-national research team developed and piloted a 4-month intervention engaging household triads (newly married women, their husbands, and mothers-in-law) toward advancing gender equity, personal agency, and reproductive health. This study evaluates the impact on family planning and fertility decision-making., Methods: In 2021, Sumadhur was piloted in six villages with 30 household triads (90 participants). Pre/post surveys of all participants were analyzed using paired sample nonparametric tests and in-depth interviews with a subset of 45 participants were transcribed and analyzed thematically., Results: Sumadhur significantly impacted (p < .05) norms related to pregnancy spacing and timing, and sex preference of children, as well as knowledge about family planning benefits, pregnancy prevention methods, and abortion legality. Family planning intent also increased among newly married women. Qualitative findings revealed improved family dynamics and gender equity, and shed light on remaining challenges., Conclusions: Entrenched social norms surrounding fertility and family planning contrasted with participants' personal beliefs, highlighting needed community-level shifts to improve reproductive health in Nepal. Engagement of influential community- and family-members is key to improving norms and reproductive health. Additionally, promising interventions such as Sumadhur should be scaled up and reassessed., (© 2023. The Author(s).)

To meet the postpartum family planning (PPFP) needs of women in Nepal, an intervention was launched to integrate PPFP counselling and postpartum IUD (PPIUD) insertion into maternity care. Women delivering in study hospitals over a period of 18 months were interviewed at the time of delivery and at 15 months following the end of the study enrollment period to assess if the impact of the intervention observed at the end of the study was maintained. Data were collected prior to the intervention, at the middle month of the intervention roll out, at the end of the enrollment period and 15 months after the end of the enrollment period. We compared PPFP counselling and insertion rates before, during, at the end of and after the intervention study period, using cross-tabulation and chi-square tests. Overall, PPFP counselling rates increased from 11% at the baseline month to 45% at the end of the enrollment in February 2017 and remained the same 15 months later in July 2018. PPIUD uptake, however, rose from a negligible 0.1% at the baseline to 4.3% in February 2017, but declined to 3.4% in July 2018. PPIUD uptake among women who were counselled showed a similar trend, increasing from 1.9% at the baseline to 9.6% in February 2017 and declining to 6.0% in July 2018. The intervention had an appreciable continued impact on PPIUD counselling rates and although PPIUD uptake rose during the intervention, this trend was not observed in the 15 months post-study follow up. The impact of the intervention was greater and persistent in hospitals that had a longer period of exposure to intervention. The results suggest that counselling was well integrated with the maternity care, though uptake of PPIUD dropped after intervention activities such as active monitoring, technical supervision, provision of IUDs and training were withdrawn. Trial registration: This study has been registered with Clinical Trial.gov. The registration number is NCT02718222. Details about the study design have been published by Canning et al, 2016., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Puri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: In Nepal, abortion is legal on request through 12 weeks of pregnancy and up to 28 weeks for health and other reasons. Abortion is available at public facilities at no cost and by trained private providers. Yet, over half of abortions are provided outside this legal system. We sought to investigate the extent to which patients are denied an abortion at clinics legally able to provide services and factors associated with presenting late for care, being denied, and receiving an abortion after being denied., Methods: We used data from a prospective longitudinal study with 1835 women aged 15-45. Between April 2019 and December 2020, we recruited 1,835 women seeking abortions at 22 sites across Nepal, including those seeking care at any gestational age (n = 537) and then only those seeking care at or after 10 weeks of gestation or do not know their gestational age (n = 1,298). We conducted interviewer-led surveys with these women at the time they were seeking abortion service (n = 1,835), at six weeks after abortion-seeking (n = 1523) and six-month intervals for three years. Using descriptive and multivariable logistic regression models, we examined factors associated with presenting for abortion before versus after 10 weeks gestation, with receiving versus being denied an abortion, and with continuing the pregnancy after being denied care. We also described reasons for the denial of care and how and where participants sought abortion care subsequent to being denied. Mixed-effects models was used to accounting clustering effect at the facility level., Results: Among those recruited when eligibility included seeking abortion at any gestational age, four in ten women sought abortion care beyond 10 weeks or did not know their gestation and just over one in ten was denied care. Of the full sample, 73% were at or beyond 10 weeks gestation, 44% were denied care, and 60% of those denied continued to seek care after denial. Nearly three-quarters of those denied care were legally eligible for abortion, based on their gestation and pre-existing conditions. Women with lower socioeconomic status, including those who were younger, less educated, and less wealthy, were more likely to present later for abortion, more likely to be turned away, and more likely to continue the pregnancy after denial of care., Conclusion: Denial of legal abortion care in Nepal is common, particularly among those with fewer resources. The majority of those denied in the sample should have been able to obtain care according to Nepal's abortion law. Abortion denial could have significant potential implications for the health and well-being of women and their families in Nepal., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Puri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Research calls for the sexual and reproductive rights field to prioritize gender norms to ensure that women can act on their reproductive rights. However, there is a gap in accepted measures. We addressed this by including important theoretical components of gender norms: differentiating between descriptive and injunctive norms and adding a referent group. Our team originally developed and validated the G-NORM, a gender norms scale, in India. In this paper, we describe how we subsequently adapted and validated it in Nepal. We administered items to women of reproductive age, conducted exploratory and confirmatory factor analysis, and examined associations between the subscales and reproductive health outcomes. Like the original G-NORM, our factor analyses showed that descriptive norms and injunctive norms comprise two distinct scales which fit the data well and had Cronbach alphas of 0.92 and 0.89. More equitable descriptive gender norms were associated with higher decision-making scores, increased odds of intending to use family planning, disagreeing that it is wrong to use family planning, and older ideal age at marriage. Injunctive gender norms were only associated with disagreeing that it is wrong to use family planning. Findings offer an improved measure of gender norms in Nepal and provide evidence that gender norms are critical for agency and reproductive health outcomes., (© 2022 The Authors. Studies in Family Planning published by Wiley Periodicals LLC on behalf of Population Council.)

Revealing the molecular events associated with reprogramming different somatic cell types to pluripotency is critical for understanding the characteristics of induced pluripotent stem cell (iPSC) therapeutic derivatives. Inducible reprogramming factor transgenic cells or animals-designated as secondary (2°) reprogramming systems-not only provide excellent experimental tools for such studies but also offer a strategy to study the variances in cellular reprogramming outcomes due to different in vitro and in vivo environments. To make such studies less cumbersome, it is desirable to have a variety of efficient reprogrammable mouse systems to induce successful mass reprogramming in somatic cell types. Here, we report the development of two transgenic mouse lines from which 2° cells reprogram with unprecedented efficiency. These systems were derived by exposing primary reprogramming cells containing doxycycline-inducible Yamanaka factor expression to a transient interruption in transgene expression, resulting in selection for a subset of clones with robust transgene response. These systems also include reporter genes enabling easy readout of endogenous Oct4 activation (GFP), indicative of pluripotency, and reprogramming transgene expression (mCherry). Notably, somatic cells derived from various fetal and adult tissues from these 2° mouse lines gave rise to highly efficient and rapid reprogramming, with transgene-independent iPSC colonies emerging as early as 1 wk after induction. These mouse lines serve as a powerful tool to explore sources of variability in reprogramming and the mechanistic underpinnings of efficient reprogramming systems.

Despite men's poorer outcomes than women in many health issues such as life expectancy, ischemic heart disease, hypertension, diabetes, HIV/AIDS, traffic-related injuries, drug and alcohol abuse, etc, Nepal's health care strategy does not have an adequately focused program to address men's health needs. This comment aims to illustrate the differences in selected health indicators between men and women and suggest Nepal's health sector strategy 2022-2030 should address to advance men's health needs. Keywords: Health policy; men's health; Nepal.

Objective: Although abortion in Nepal is broadly legal and free of charge, many women seek abortion care outside the legal system, including from pharmacies. We evaluated the prevalence of, and factors associated with, prior unsuccessful abortion attempts among women presenting to 14 randomly-selected government approved abortion health facilities across Nepal., Methods: Eligible participants were recruited in 2019 by trained research staff from certified abortion facilities. Participants (n = 1160) completed research staff-administered baseline surveys. We used multivariable mixed-effects logistic regression models to evaluate factors associated with having attempted pharmacy abortion prior to coming to the health facility., Results: Almost one in seven (14%) women had tried to end their pregnancy before presenting to a participating clinic, often (9%) using medication obtained from a pharmacy. Women who lived farther from the clinic (aOR 1.28 per log hours travel time, 95% CI 1.10-1.49) and who reported financial difficulty in accessing the clinic (19% vs. 10%, aOR 2.10, 95% CI 1.20-3.70) had increased odds of having tried to access abortion through a pharmacy., Conclusion: Integrating pharmacies into the legal network of abortion providers may improve access to safe care, particularly for rural women with financial and practical travel limitations., (© 2022 International Federation of Gynecology and Obstetrics.)

Background: Postpartum women have high rates of unmet need for modern contraception in the two years following birth in Nepal. We assessed whether providing contraceptive counseling during pregnancy and/or prior to discharge from the hospital for birth or after discharge from the hospital for birth was associated with reduced postpartum unmet need in Nepal., Methods: We used data from a larger a stepped-wedge, cluster randomized trial, including contraceptive counselling in six tertiary hospitals. Group 1 hospitals (three hospitals) initiated the intervention after three months of baseline data collection, while Group 2 hospitals (three hospitals) initiated the same intervention after nine months. We have enrolled 21,280 women in the baseline interviews and conducted two follow-up interviews with them, one and two years after they had delivered in one of our study hospitals. We estimated the effect of counseling and its timing (pre-discharge, post- discharge, both, or neither) on unmet need for modern contraception in the postpartum period, using random-effects logistic regressions., Results: Unmet need for modern contraception was high (54% at one year and 50% at two years). Women counseled in either the pre-discharge period (Odds ratio [OR] 0·86; 95% CI: 0·80, 0·93) or in the post-discharge period (OR 0·86; 95% CI: 0·79, 0·93) were less likely to have an unmet need in the postpartum period compared to women with no counseling. However, women who received counseling in both the pre- and post-discharge period were 27% less likely than women who had not received counseling to have unmet need (OR 0.73; 95% CI: 0·67, 0·80)., Conclusions: Counseling women either before or after discharge reduces unmet need for postpartum contraception but counseling in both periods is most effective., Competing Interests: The authors have declared that no competing interests exist.

Background: Health service providers play a key role in addressing women's need for postpartum pregnancy prevention. Yet, in Nepal, little is known about providers' knowledge, attitudes, and practice (KAP) on providing postpartum family planning (PPFP), particularly the immediate postpartum intrauterine device (PPIUD). This paper assesses providers KAP towards the provision of PPIUDs in Nepal prior to a PPIUD intervention to gain a baseline insight and analyzes whether their KAP changes both 6 and 24 months after the start of the intervention., Methods: Data come from a randomized trial assessing the impact of a PPIUD intervention in Nepal between 2015 and 2017. We interviewed 96 providers working in six study hospitals who completed a baseline interview and follow-up interviews at 6 and 24 months. We used descriptive analysis, McNemar's test and the Wilcoxon signed-rank test to assess KAP of providers over 2 years., Results: The PPIUD KAP scores improved significantly between the baseline and 6-month follow-up. Knowledge scores increased from 2.9 out of 4 to 3.5, attitude scores increased from 4 out of 7 to 5.3, and practice scores increased from 0.9 out of 3 to 2.8. There was a significant increase in positive attitude and practice between 6 and 24 months. Knowledge on a women's chance of getting pregnant while using an IUD was poor. Attitudes on recommending a PPIUD to different women significantly improved, however, attitudes towards recommending a PPIUD to unmarried women and women who have had an ectopic pregnancy improved the least. Practice of PPIUD counseling and insertion improved significantly from baseline to 24 months, from 10.4 and 9.4% to 99% respectively., Conclusions: Although KAP improved significantly among providers during the PPIUD intervention, providers' knowledge on a women's chance of getting pregnant while using an IUD and attitudes towards recommending a PPIUD to unmarried women and women who have had an ectopic pregnancy improved the least. Provider KAP could be improved further through ongoing and more in-depth training to maintain providers' knowledge, reduce provider bias and misconceptions about PPIUD eligibility, and to ensure providers understand the importance of birth spacing.

Context: Providers' and women's characteristics are associated with postpartum copper IUD (PPIUD) outcomes, but the relationship between providers' level of experience and PPIUD expulsion and discontinuation has not been established., Methods: Data on 1,232 women and 118 providers who took part in a randomized trial of a PPIUD counselling and provision intervention in Nepal between 2015 and 2017 were used to identify associations between providers' and women's characteristics and PPIUD outcomes. Multinomial logistic regression models were used to estimate PPIUD expulsion and discontinuation risks at two years after insertion., Results: Thirteen percent of women had had partial or complete expulsions and 29% had discontinued PPIUD use by two years. Having a provider who had done at least 10 previous insertions was associated with lower risk of expulsion rather than continuation (relative risk ratio, 0.5) relative to having a less-experienced provider. Women had a higher risk of both expulsion and discontinuation relative to continuation if they were younger than 21 rather than aged 26-30 (2.4 and 1.7, respectively) or if they belonged to the Dalit rather than Brahmin caste (2.2 and 1.9, respectively). Women whose husbands did not live at home also had elevated discontinuation risks., Conclusion: The findings highlight the need for increased training and supervision of providers during their first 10 PPIUD insertions. Counselling on risk of expulsion may especially benefit younger and Dalit women, and should include partners and other family members to avoid any stigma surrounding PPIUD use by women whose partner is away from home for a prolonged period.

The national lockdown imposed in Nepal as a response to the COVID-19 pandemic is having indirect consequences on sexual and reproductive (SRH) in Nepal. Although the Government of Nepal and partners have committed to ensuring the continuity of SRH services during the pandemic, this comment aims to illustrate the potential impacts to SRH if these commitments are not met. Keywords: COVID-19; Nepal; reproductive health; sexual health.

Objectives: Nationally representative evidence on abortion service provision is scarce in South Asia. To inform improvements in service provision, this paper assesses the availability of facility-based postabortion services in Nepal, India (six states), Bangladesh and Pakistan, and legal abortion services in India and Nepal and Bangladesh (where the official term used is menstrual regulation or MR)., Study Design: The paper presents comparable indicators on three aspects of abortion service provision from representative surveys of public and private sector facilities, conducted over 2012-2015. Indicators cover three areas: (a) need for abortion-related care (total number of abortions and percent of abortions that are legal and the postabortion treatment rate); (b) availability and accessibility of facility-based abortion-related services (percent of facilities offering only one of the two services, percent which are public and percent located in rural areas); (c) quality of facility-based abortion care (percent of legal abortions using procedures not recommended by WHO and percent of women turned away when seeking abortion or MR services)., Results: The proportion of all abortions that are illegal ranges from 58% to almost 78% in the three countries where abortion is permitted under broad criteria. The annual treatment rate for abortion complications ranges from about 4 to 26 per 1000 women ages 15-49 across the countries and states covered. In India and Nepal, less than 40% of public sector facilities that are permitted to provide abortion services do so; in Bangladesh, the situation is somewhat better, at 53% providing MR. Across the six Indian states, 4-43% of facilities that offer abortion care are located in rural areas, disproportionately lower than the proportion of women living in rural areas (49-87%). About 30-60% of facilities offered only postabortion care and did not offer legal services in the three countries where legal services are permitted (with the sole exception of Tamil Nadu where this proportion was only 11%); of the remaining facilities, the large majority offered both services. Medication abortion is offered by the large majority of facilities that provide induced abortion and accounts for 40-45%, of facility-based abortions in Nepal and four of the states of India; in Assam and Bihar, this proportion was much lower (13% and 27% respectively). Invasive procedures that are not recommended by WHO are more widely used in India (up to 25-37% of facility-based abortions are D&C procedures; the large majority of this group are D&C, and a small proportion may be D&E, a WHO-recommended abortion procedure, that could not be separated out in this study because providers use the two labels interchangeably); by comparison, the proportion is much smaller in Nepal (5%). Between 22% to a little over half of facilities turned away some women who would otherwise be eligible for an abortion or MR procedure in Nepal, the six Indian states, and Bangladesh., Conclusions: There is an urgent need to increase access to abortion, MR and postabortion services, especially for rural women. Greater access to legal abortion/MR services in the three countries that permit these procedures would increase the proportion of abortions that are legal and safe, reduce morbidity and the need for facility-based treatment for complications. Broadening the legal criteria under which abortion is permitted in Pakistan, and implementing access under such broader criteria, is needed to achieve the same improvements in Pakistan. Ensuring that these services are of high quality and comprehensive-meeting WHO-recommended standards-is essential to protect women's reproductive health and rights., Implications: To improve access to abortion, MR and postabortion care in South Asia, all facilities (public and private) permitted to provide these services should do so, and should include medication abortion. Improvements in quality of care are critical: invasive procedures (D&C) should be eliminated through adherence to WHO's standards of safe abortion care and women seeking abortions should not be turned away because of providers' biases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Postpartum intrauterine device (PPIUD) use remains very low in Nepal despite high levels of unmet need for postpartum family planning and the national government's efforts to promote its use. This study investigates reasons for continuing or discontinuing PPIUD use among Nepali women., Methods: We conducted in-depth interviews (IDIs) with 13 women who had discontinued PPIUD use and 12 women who were continuing to use the method 9 months or longer following the insertion. All interviews were audio recorded, transcribed, translated into English, and analyzed using a thematic approach., Results: Women discontinued PPIUD for several reasons: 1) side effects such as excessive bleeding during menstruation, nausea, back and abdominal pain; 2) poor quality of counselling and, relatedly, mismatched expectations in terms of device use; and 3) lack of family support from husbands and in-laws. In contrast, women who were continuing to use the method at the time of the study stated that they had not experienced side-effects, had received appropriate information during counselling sessions, and had the backing of their family members in terms of using PPIUD., Conclusion: Experiencing side-effects or complications following PPIUD insertion and poor quality of family planning counselling were the two main reasons for discontinuation. Family members appeared to play a major role in influencing a woman's decision to continue or discontinue PPIUD suggesting that counseling may need to be expanded to them as well. Improving quality of counselling by providing complete and balanced information of family planning methods as well as ensuring sufficient time for counselling and extending PPIUD service availability at lower level clinics/health posts will potentially increase the uptake and continued use of postpartum family planning, including PPIUD, in Nepal.

Background: Though modern contraceptive use among married women in Nepal has increased from 26% in 1996 to 43% in 2016, it remains low among postpartum women. Integration of counselling on family planning (FP) at the time of antenatal care (ANC) and delivery has the potential to increase post-partum contraceptive use. This study investigates the quality of FP counselling services provided during ANC visits and women's perceptions of its effectiveness in assisting them to make a post-partum family planning (PPFP) decision., Methods: In-depth interviews (IDIs) were conducted with 24 pregnant women who had attended at least two ANC visits in one of the six public hospitals that had received an intervention that sought to integrate FP counselling in maternity care services and introduce postpartum intrauterine device insertion in the immediate postpartum period. IDIs data were collected as part of a process evaluation of this intervention. Women were selected using maximum variation sampling to represent different socio-demographic characteristics. IDIs were audio recorded, transcribed verbatim in Nepali, and translated into English. Data were organized using Bruce-Jain quality of care framework and analyzed thematically., Results: Overall, the quality of FP counselling during ANC was unsatisfactory based on patient expectations and experience of interactions with providers, as well as FP methods offered. Despite their interest, most women reported that they did not receive thorough information about FP, and about a third of them said that they did not receive any counselling services on PPFP. Reasons for dissatisfaction with counselling services included very crowded environment, short time with the provider, non-availability of provider, long waiting times, limited number of days for ANC services, and lack of comprehensive FP-related information, education and counselling (IEC) materials. Women visiting hospitals with a dedicated FP counselor reported higher quality of FP counselling., Conclusions: There is an urgent need to re-visit the format of counselling on PPFP during ANC visits, corresponding IEC materials, counselling setting, and to strengthen availability and interaction with providers in order to improve quality, experience and satisfaction with FP counselling during ANC visits. Improvements in infrastructure and human resources are also needed to adequately meet women's needs.

Despite the legalization of abortion in 2002 has made extensive efforts to expand services throughout the country, access to safe abortion care remains a constraint in Nepal, particularly in remote areas where trained providers and equipment are scarce. Expanding access to medical abortion (MA) through pharmacy workers could be a promising avenue to reach such women with safe and convenient care, but neither Nepali law nor the World Health Organization (WHO) recommends pharmacy provision of MA citing the reason of insufficient evidence. This paper examines the pharmacy provision of MA in expanding women's access to safe MA service in Nepal. Available literature between 2005 and 2018 was searched, and relevant information was extracted using a template, and findings were summarized and interpreted according to the objective of this study. We identified 83 studies that were related to MA in Nepal, but only five of them reported about pharmacy workers' involvement in the provision of MA in Nepal. Findings suggest that trained pharmacy workers can safely and effectively provide MA. MA services provided by pharmacy workers are acceptable to women who were satisfied with the service they had received. Therefore, it is essential that the Government of Nepal acknowledges the role pharmacy workers currently play in the provision of MA and formulate polices to permit pharmacy workers to give information about and dispense MA tablets to women in the first trimester of pregnancy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The ability to generate induced pluripotent stem cells from differentiated cell types has enabled researchers to engineer cell states. Although studies have identified molecular networks that reprogram cells to pluripotency, the cellular dynamics of these processes remain poorly understood. Here, by combining cellular barcoding, mathematical modeling, and lineage tracing approaches, we demonstrate that reprogramming dynamics in heterogeneous populations are driven by dominant "elite" clones. Clones arise a priori from a population of poised mouse embryonic fibroblasts derived from Wnt1-expressing cells that may represent a neural crest-derived population. This work highlights the importance of cellular dynamics in fate programming outcomes and uncovers cell competition as a mechanism by which cells with eliteness emerge to occupy and dominate the reprogramming niche., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Health service providers play a key role in addressing women's need for pregnancy prevention, especially during the postpartum period. Yet, in Nepal, little is known about their views on providing postpartum family planning (PPFP) services and postpartum contraceptive methods such as immediate postpartum intra-uterine devices (PPIUD). This paper explores the perspectives of different types of providers on PPFP including PPIUD, their confidence in providing PPFP services, and their willingness to share their knowledge and skills with colleagues after receiving PPFP and PPIUD training., Methods: In-depth interviews were conducted with 14 obstetricians/gynecologists and nurses from six tertiary level public hospitals in Nepal after they received PPFP and PPIUD training as part of an intervention aimed at integrating PPFP counseling and insertion into routine maternity care services. The interviews were audio recorded, transcribed, and analyzed using a thematic approach., Results: Providers identified several advantages of PPFP, supported the provision of such services, and were willing to transfer their newly acquired skills to colleagues in other facilities who had not received PPFP and PPIUD training. However, many providers identified several supply-side and training-related barriers to providing high quality PPFP services, such as, (i) lack of adequate human resources, particularly a FP counselor; (ii) work overload; (iii) lack of private space for counseling; (iv) lack of IUDs and information, education and counseling materials; and (v) lack of support from hospital management., Conclusions: Providers appeared to be motivated to deliver quality PPFP services and transfer their knowledge to colleagues but identified several barriers which prevented them from doing so. Future efforts to improve provision of quality PPFP services should address the barriers identified by providers.

Objective: To examine whether auxiliary nurse-midwife provision of medical abortion in pharmacies was associated with reduced post-abortion contraceptive use in Nepal., Methods: The present prospective observational study compared contraceptive use among women aged 16-45 years and up to 63 days of pregnancy, who presented at one of six privately-owned pharmacies or six public health facilities in the Chitwan and Jhapa districts of Nepal for medical abortion between October 16, 2014, and September 1, 2015. Participants obtained medical abortions per Nepali protocol and completed a follow-up visit and interview at 14-21 days. Effective contraceptive use was compared between abortion care settings using multivariable mixed effects logistic regression., Results: Of 605 participants, 600 completed follow-up at 14-21 days; 474 (79.0%) were using a contraceptive method, most commonly pills (180 [30.0%]) and injectables (175 [29.2%]), followed by condoms (82 [13.7%]), long-acting reversible methods (33 [5.5%]), and sterilization (4 [0.7%]). Receipt of care from a private pharmacy was not associated with a difference in the use of hormonal or long-acting methods (adjusted odd ratio 0.89, 95% confidence interval 0.60-1.33)., Conclusion: Medical abortion provision from pharmacies by qualified providers can provide women with necessary induced-abortion care while not compromising longer-term pregnancy prevention., (© 2018 International Federation of Gynecology and Obstetrics.)

Background: Despite legalization of abortion in Nepal in 2002, many women are still unable to access legal services. This paper examines providers' views, experiences with abortion denial, and knowledge related to abortion provision, and identifies areas for improvement in quality of care., Methods: We conducted a structured survey with 106 abortion care providers at 55 government-approved safe abortion facilities across five districts of Nepal in 2017. We assessed reasons for denial of abortion care, knowledge about laws, barriers to provision and attitudes towards abortion., Results: Almost all providers (96%) reported that they have ever refused clients for abortion services. Common reasons included beyond 12 weeks gestation (93%), sex selective abortion (86%), and medical contraindications (85%). One in four providers denied abortion for lack of drugs or trained personnel, and one third denied services when they perceived that the woman's reasons for abortion were insufficient. Only a third of providers knew all three legal indications for abortion -- less than or equal to 12 weeks of pregnancy on request, up to 18 weeks for rape or incest, and any time for maternal or fetal health risk. Overall, providers were in favor of legal abortion but a substantial proportion had mixed or negative attitudes about the service., Conclusions: Improvements in training to address providers' inadequate knowledge about the abortion law may reduce inappropriate denial of abortion. Establishing referral networks in the case of abortion denial and ensuring regular supply of medical abortion drugs would help more women access abortion care in Nepal.

Cellular reprogramming remodels the gene expression program by re-setting the epigenome of somatic cells into an embryonic-like pluripotent state. Post-translational modifications of histones play an important role in this process. Previously, we found by ChIP-seq widespread changes of specific histone H3 marks in two divergent reprogramming routes leading to alternative pluripotent sates . Here, using an unbiased middle-down proteomics approach we have identified 72 unique isoforms of histone H4 and quantified 56 of them in the same set of samples. We found substantial differences between somatic and late-phase reprogramming cells. Also, ESCs and iPSCs displayed higher levels of H4 acetylation and tri-methylation concomitantly with lower levels of mono- and di-methylation when compared to cells undergoing reprogramming. Our data shows that the epigenetic remodeling induced by the reprogramming process goes beyond histone H3 and reveals the importance of H4 modifications as well. The presented data is a valuable resource to study the epigenetic mechanisms involved in the acquisition of induced pluripotency. All MS data have been deposited in the ProteomeXchange with identifier PXD002062 (http://proteomecentral.proteomexchange.org/dataset/PXD002062)., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture.

Development of the cardiovascular system is critically dependent on the ability of endothelial cells (ECs) to reorganize their intracellular actin architecture to facilitate migration, adhesion, and morphogenesis. Nck family cytoskeletal adaptors function as key mediators of actin dynamics in numerous cell types, though their role in EC biology remains largely unexplored. Here, we demonstrate an essential requirement for Nck within ECs. Mouse embryos lacking endothelial Nck1/2 expression develop extensive angiogenic defects that result in lethality at about embryonic day 10. Mutant embryos show immature vascular networks, with decreased vessel branching, aberrant perivascular cell recruitment, and reduced cardiac trabeculation. Strikingly, embryos deficient in endothelial Nck also fail to undergo the endothelial-to-mesenchymal transition (EnMT) required for cardiac valve morphogenesis, with loss of Nck disrupting expression of major EnMT markers, as well as suppressing mesenchymal outgrowth. Furthermore, we show that Nck-null ECs are unable to migrate downstream of vascular endothelial growth factor and angiopoietin-1, and they exhibit profound perturbations in cytoskeletal patterning, with disorganized cellular projections, impaired focal adhesion turnover, and disrupted actin-based signaling. Our collective findings thereby reveal a crucial role for Nck as a master regulator within the endothelium to control actin cytoskeleton organization, vascular network remodeling, and EnMT during cardiovascular development., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or αvβ3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Targeted ultrasound contrast imaging has the potential to become a reliable molecular imaging tool. A better understanding of the quantitative aspects of molecular ultrasound technology could facilitate the translation of this technique to the clinic for the purposes of assessing vascular pathology and detecting individual response to treatment. The objective of this study was to evaluate whether targeted ultrasound contrast-enhanced imaging can provide a quantitative measure of endogenous biomarkers. Endoglin, an endothelial biomarker involved in the processes of development, vascular homeostasis, and altered in diseases, including hereditary hemorrhagic telangiectasia type 1 and tumor angiogenesis, was the selected target. We used a parallel plate perfusion chamber in which endoglin-targeted (MBE), rat isotype IgG2 control and untargeted microbubbles were perfused across endoglin wild-type (Eng+/+), heterozygous (Eng+/-) and null (Eng-/-) embryonic mouse endothelial cells and their adhesion quantified. Microbubble binding was also assessed in late-gestation, isolated living transgenic Eng+/- and Eng+/+ embryos. Nonlinear contrast-specific ultrasound imaging performed at 21 MHz was used to collect contrast mean power ratios for all bubble types. Statistically significant differences in microbubble binding were found across genotypes for both in vitro (p<0.05) and embryonic studies (p<0.001); MBE binding was approximately twofold higher in Eng+/+ cells and embryos compared with their Eng+/- counterparts. These results suggest that molecular ultrasound is capable of reliably differentiating between molecular genotypes and relating receptor densities to quantifiable molecular ultrasound levels.

Somatic cell reprogramming to a pluripotent state continues to challenge many of our assumptions about cellular specification, and despite major efforts, we lack a complete molecular characterization of the reprograming process. To address this gap in knowledge, we generated extensive transcriptomic, epigenomic and proteomic data sets describing the reprogramming routes leading from mouse embryonic fibroblasts to induced pluripotency. Through integrative analysis, we reveal that cells transition through distinct gene expression and epigenetic signatures and bifurcate towards reprogramming transgene-dependent and -independent stable pluripotent states. Early transcriptional events, driven by high levels of reprogramming transcription factor expression, are associated with widespread loss of histone H3 lysine 27 (H3K27me3) trimethylation, representing a general opening of the chromatin state. Maintenance of high transgene levels leads to re-acquisition of H3K27me3 and a stable pluripotent state that is alternative to the embryonic stem cell (ESC)-like fate. Lowering transgene levels at an intermediate phase, however, guides the process to the acquisition of ESC-like chromatin and DNA methylation signature. Our data provide a comprehensive molecular description of the reprogramming routes and is accessible through the Project Grandiose portal at http://www.stemformatics.org.

Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.

The ectopic expression of Oct4, Klf4, c-Myc and Sox2 (OKMS) transcription factors allows reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). The reprogramming process, which involves a complex network of molecular events, is not yet fully characterized. Here we perform a quantitative mass spectrometry-based analysis to probe in-depth dynamic proteome changes during somatic cell reprogramming. Our data reveal defined waves of proteome resetting, with the first wave occurring 48 h after the activation of the reprogramming transgenes and involving specific biological processes linked to the c-Myc transcriptional network. A second wave of proteome reorganization occurs in a later stage of reprogramming, where we characterize the proteome of two distinct pluripotent cellular populations. In addition, the overlay of our proteome resource with parallel generated -omics data is explored to identify post-transcriptionally regulated proteins involved in key steps during reprogramming.

Reprogramming of somatic cells to induced pluripotent stem cells involves a dynamic rearrangement of the epigenetic landscape. To characterize this epigenomic roadmap, we have performed MethylC-seq, ChIP-seq (H3K4/K27/K36me3) and RNA-Seq on samples taken at several time points during murine secondary reprogramming as part of Project Grandiose. We find that DNA methylation gain during reprogramming occurs gradually, while loss is achieved only at the ESC-like state. Binding sites of activated factors exhibit focal demethylation during reprogramming, while ESC-like pluripotent cells are distinguished by extension of demethylation to the wider neighbourhood. We observed that genes with CpG-rich promoters demonstrate stable low methylation and strong engagement of histone marks, whereas genes with CpG-poor promoters are safeguarded by methylation. Such DNA methylation-driven control is the key to the regulation of ESC-pluripotency genes, including Dppa4, Dppa5a and Esrrb. These results reveal the crucial role that DNA methylation plays as an epigenetic switch driving somatic cells to pluripotency.

MicroRNAs (miRNAs) are critical to somatic cell reprogramming into induced pluripotent stem cells (iPSCs), however, exactly how miRNA expression changes support the transition to pluripotency requires further investigation. Here we use a murine secondary reprogramming system to sample cellular trajectories towards iPSCs or a novel pluripotent 'F-class' state and perform small RNA sequencing. We detect sweeping changes in an early and a late wave, revealing that distinct miRNA milieus characterize alternate states of pluripotency. miRNA isoform expression is common but surprisingly varies little between cell states. Referencing other omic data sets generated in parallel, we find that miRNA expression is changed through transcriptional and post-transcriptional mechanisms. miRNA transcription is commonly regulated by dynamic histone modification, while DNA methylation/demethylation consolidates these changes at multiple loci. Importantly, our results suggest that a novel subset of distinctly expressed miRNAs supports pluripotency in the F-class state, substituting for miRNAs that serve such roles in iPSCs.

As a tumor surrogate, the mouse embryo presents as an excellent alternative for examining the binding of angiogenesis-targeting microbubbles and assessing the quantitative nature of molecular ultrasound. We establish the validity of this model by developing a robust method to study microbubble kinetic behavior and investigate the reproducibility of targeted binding in the murine embryo. Vascular endothelial growth factor receptor 2 (VEGFR2)-targeted (MBV), rat immunoglobulin G2 (IgG2) control antibody-targeted (MBC) and untargeted (MBU) microbubbles were introduced into vasculature of living mouse embryos. Non-linear contrast-specific and B-mode ultrasound imaging, performed at 21 MHz with a Vevo-2100 scanner, was used to collect basic perfusion parameters and contrast mean power ratios for all bubble types. We observed a twofold increase (p < 0.001) in contrast mean power ratios for MBV (4.14 ± 1.78) compared with those for MBC (1.95 ± 0.78) and MBU (1.79 ± 0.45). Targeted imaging of endogenous endothelial cell surface markers in mouse embryos is possible with labeled microbubbles. The mouse embryo thus presents as a versatile model for testing the performance of ultrasound molecular targeting, where further development of quantitative imaging techniques may enable rapid evaluations of biomarker expression in studies of vascular development, disease and angiogenesis., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1(+)CD31(dim)vascular endothelial (VE)-cadherin(-)CD45(-) precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1(+) precursor cell, but not the VE-cadherin(+) endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1(+) precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1(+) local precursor to VSMC in a spatiotemporal fashion across all vascular beds.

Extraordinary advances in pluripotent stem cell research have initiated an era of hope for regenerative strategies to treat human disease. Besides embryonic stem cells, the discovery of induced pluripotent stem cells widened the possibility of patient-specific cell therapy, drug discovery, and disease modeling. Although similar, it has become clear that these two pluripotent cell types display significant differences. In this review, we explore current knowledge of the molecular and functional similarities and differences between these two cell types to emphasize the necessity for thorough characterization of their properties as well as their differentiation capabilities in the pluripotent state. Such comparative studies will be crucial for determining the more suitable cell type for future stem cell-based therapies for human degenerative diseases., (Copyright © 2011 AlphaMed Press.)

Vascular patterning depends on precisely coordinated timing of endothelial cell differentiation and onset of cardiac function. Endoglin is a transmembrane receptor for members of the TGF-beta superfamily that is expressed on endothelial cells from early embryonic gestation to adult life. Heterozygous loss of function mutations in human ENDOGLIN cause Hereditary Hemorrhagic Telangiectasia Type 1, a vascular disorder characterized by arteriovenous malformations that lead to hemorrhage and stroke. Endoglin null mice die in embryogenesis with numerous lesions in the cardiovascular tree including incomplete yolk sac vessel branching and remodeling, vessel dilation, hemorrhage and abnormal cardiac morphogenesis. Since defects in multiple cardiovascular tissues confound interpretations of these observations, we performed in vivo chimeric rescue analysis using Endoglin null embryonic stem cells. We demonstrate that Endoglin is required cell autonomously for endocardial to mesenchymal transition during formation of the endocardial cushions. Endoglin null cells contribute widely to endothelium in chimeric embryos rescued from cardiac development defects, indicating that Endoglin is dispensable for angiogenesis and vascular remodeling in the midgestation embryo, but is required for early patterning of the heart.

The development of the cardiovascular system and the development of the early hematopoietic systems are closely related, and both require signaling through the Tie2 receptor tyrosine kinase. Although endothelial cells and hematopoietic cells as well as their precursors share common gene expression patterns during development, it remains completely unknown how Tie2 signaling coordinately regulates cardiovascular development and early hematopoiesis in vivo. We show here that mice with a targeted mutation in tyrosine residue 1100 in the carboxyl-terminal tail of Tie2 display defective cardiac development and impaired hematopoietic and endothelial cell development in the paraaortic splanchnopleural mesoderm similar to that seen in Tie2-null mutant mice. Surprisingly, however, unlike Tie2-null mutant mice, mice deficient in signaling through this tyrosine residue show a normal association of perivascular cells with nascent blood vessels. These studies are the first to demonstrate the physiological importance of a single tyrosine residue in Tie2, and they suggest that multiple tyrosine residues in the receptor may coordinate cardiovascular development and early hematopoietic development.

In mammals, the continuous production of hematopoietic cells (HCs) is sustained by a small number of hematopoietic stem cells (HSCs) residing in the bone marrow. Early HSC activity arises in the aorta-gonad mesonephros region, within cells localized to the ventral floor of the major blood vessels, suggesting that the first HSCs may be derived from cells capable of giving rise to the hematopoietic system and to the endothelial cells of the vasculature. TIE1 (TIE) and TIE2 (TEK) are related receptor tyrosine kinases with an embryonic expression pattern in endothelial cells, their precursors, and HCs, suggestive of a role in the divergence and function of both lineages. Indeed, gene targeting approaches have shown that TIE1, TIE2, and ligands for TIE2, the angiopoietins, are essential for vascular development and maintenance. To explore possible roles for these receptors in HCs, we have examined the ability of embryonic cells lacking both TIE1 and TIE2 to contribute to developmental and adult hematopoiesis by generating chimeric animals between normal embryonic cells and cells lacking these receptors. We show here that TIE receptors are not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo and fetus; surprisingly, however, these receptors are specifically required during postnatal bone marrow hematopoiesis.

The molecular mechanisms responsible for organ-specific differences in vascular development are not well established. Animals lacking the receptor tyrosine kinase Tie1 die of hemorrhage and pulmonary edema. Furthermore, cells lacking Tie1 are excluded from blood vessels of the mature lung. These findings suggest the importance of Tie1 in the pulmonary vasculature. We quantified the organ-specific expression of Tie1 during embryonic and postnatal murine development using both quantitative real-time polymerase chain reaction (PCR) and chemiluminescence employing a tie1.lacZ reporter. In the lung, Tie1 expression increases markedly immediately prior to birth and rises further in the newborn animal, a pattern not found in other organs. Furthermore, expression of Tie1 in the lung is also unique by its persistent increase in the adult animal. This unique pattern of Tie1 gene expression in the embryonic and mature lung supports a distinct role for Tie1 in the development and function of the pulmonary vasculature.

TEK (TIE2) and TIE (TIE1) are structurally related receptor tyrosine kinases expressed in endothelial cells and their precursors. Genetic studies in the mouse have revealed essential functions of both receptors in angiogenic expansion of the vasculature during development. As previously shown, mouse embryos homozygous for a disrupted Tek allele die by day 10.5 of embryogenesis due to endocardial defects, hemorrhaging, and impaired vascular network formation. Furthermore, TIE is required cell autonomously for endothelial cell survival and extension of the vascular network during late embryogenesis. Here we have investigated possible redundancy in the TEK and TIE signalling pathways during vascular development. Vasculogenesis proceeds normally in embryos lacking both TEK and TIE, although such embryos die early in gestation of multiple cardiovascular defects. Mosaic analysis revealed an absolute requirement for TEK in the endocardium at E10.5, whereas TEK and TIE are dispensable for the initial assembly of the rest of the vasculature. In contrast, both receptors are required in the microvasculature during late organogenesis and in essentially all blood vessels of the adult. This analysis demonstrates essential functions for TEK and TIE in maintaining the integrity of the mature vasculature.

TIE is a receptor tyrosine kinase expressed in both mature endothelial cells and their precursors, as well as in some hematopoietic cells. Mouse embryos homozygous for a disrupted Tie allele die at midgestation due to impaired endothelial cell integrity and resulting hemorrhage. Here we have performed chimeric analysis to study further the function of the murine TIE in the development of embryonic vasculature and in the hematopoietic system. Cells lacking a functional Tie gene (tie(lcz)/tie(lczn-) cells) contributed to the embryonic vasculature at E10.5 as efficiently as cells heterozygous for a targeted Tie allele (tie(lcz)/+ cells). Thus, TIE does not play a significant role in vasculogenesis or in early angiogenic processes, such as formation of the intersomitic arteries and limb bud vascularization. At E15.5 tie(lcz)/tie(lczn-) cells still readily contributed to major blood vessels and to endothelial cells of organs such as lung and heart, which have been suggested to be vascularized by angioblast differentiation. In contrast, the tie(lcz)/tie(lczn-) cells were selected against in the capillary plexuses of several angiogenically vascularized tissues, such as brain and kidney. Our results thus support a role for TIE in late phases of angiogenesis but not vasculogenesis. Furthermore, the results suggest that different mechanisms regulate early and late angiogenesis and provide support for a model of differential organ vascularization by vasculogenic or angiogenic processes. Analysis of adult chimeras suggested that TIE is required to support the survival or proliferation of certain types of endothelial cells demonstrating heterogeneity in the growth/survival factor requirements in various endothelial cell populations. Chimeric analysis of adult hematopoietic cell populations, including peripheral platelets and bone marrow progenitor cells, revealed that tie(lcz)/tie(lczn-) cells were able to contribute to these cell types in a way indistinguishable from tie(lcz)/+ or wild-type cells. Thus, the primary function of TIE appears to be restricted to the endothelial cell lineage.

Vascular endothelial cells are critical for the development and function of the mammalian circulatory system. We have analyzed the role of the endothelial cell-specific receptor tyrosine kinase TIE in the mouse vasculature. Mouse embryos homozygous for a disrupted Tie allele developed severe edema, their microvasculature was ruptured and they died between days 13.5 and 14.5 of gestation. The major blood vessels of the homozygous embryos appeared normal. Cells lacking a functional Tie gene were unable to contribute to the adult kidney endothelium in chimeric animals, further demonstrating the intrinsic requirement for TIE in endothelial cells. We conclude that TIE is required during embryonic development for the integrity and survival of vascular endothelial cells, particularly in the regions undergoing angiogenic growth of capillaries. TIE is not essential, however, for vasculogenesis, the early differentiation of endothelial cells.

The gene encoding p120-rasGAP, a negative regulator of Ras, has been disrupted in mice. This Gap mutation affects the ability of endothelial cells to organize into a highly vascularized network and results in extensive neuronal cell death. Mutati ons in the Gap and Nf1 genes have a synergistic effect, such that embryos homozygous for mutations in both genes show an exacerbated Gap phenotype. Thus rasGAP and neurofibromin act together to regulate Ras activity during embryonic development.

We report the detailed developmental expression profiles of three endothelial specific receptor tyrosine kinases (RTKs) flk-1, tek, tie, as well as vascular endothelial growth factor (VEGF), the flk-1 ligand. We also examined the expression of the other VEGF receptor, flt-1, during placental development. flk-1, tek, and tie transcripts were detected sequentially at one-half day intervals starting at E7.0, suggesting that each of these RTKs play a unique role during vascularization of the mouse embryo. All three RTKs were expressed in the extraembryonic and embryonic mesoderm in regions that eventually give rise to the vasculature. Except for the expression of tek and flk-1 in the mesoderm of the amnion, the expression of these RTKs from E8.5 onwards was virtually indistinguishable. An abundant amount of flt-1 transcripts was found in the spongiotrophoblast cells of the developing placenta from E8.0 onwards. This cellular compartment is located between the maternal and labyrinthine layers of the placenta, which both express VEGF. VEGF transcripts were detected as early as E7.0 in the endoderm juxtaposed to the flk-1 positive mesoderm, and later in development VEGF expression displayed an expression profile both contiguous with that of flk-1, and also in tissues found some distance from the flk-1-expressing endothelium. These results suggest a possible dual role for VEGF which includes a chemotactic and/or a cellular maintenance role for VEGF during vascularization of the mouse embryo.

Lens-specific expression of the mouse gamma-F-crystallin gene is determined, at least in part, by a 23-bp DNA element, the gamma F-1-binding motif, located in the promoter region of the gene. To characterize the transcription factors that regulate gamma F-crystallin gene expression through this element, we have isolated three chicken cDNAs that encode proteins capable of binding specifically to the gamma F-1-binding motif. These three cDNAs represent differential splicing products from a single gene, gamma FBP. The protein isoforms encoded by two of these cDNAs differ in their ability to modulate the activity of promoters containing the gamma F-1-binding motif. Among them, gamma FBP-B functions as a transcriptional repressor in lens cells, and it's expression is developmentally regulated during lens development, suggesting a role for this isoform in the spatial regulation of gamma F-crystallin gene expression. We also show that expression of the different mRNA transcripts are differentially regulated in various tissues. Furthermore, gamma FBP transcripts are highly expressed in presomitic mesoderm and then over the entire epithelial somite. During somitic differentiation, gamma FBP expression becomes restricted to the sclerotome. These expression patterns suggest a regulatory role for the gamma FBP isoforms in sclerotome specification and/or differentiation.