Sex Reversal in Zebrafish fancl Mutants is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA-repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination

Sex Reversal in Zebrafish fancl Mutants Is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination

Mapping geographical inequalities in access to drinking water and sanitation facilities in low-income and middle-income countries, 2000-17

Background: Universal access to safe drinking water and sanitation facilities is an essential human right, recognised in the Sustainable Development Goals as crucial for preventing disease and improving human wellbeing. Comprehensive, high-resolution estimates are important to inform progress towards achieving this goal. We aimed to produce high-resolution geospatial estimates of access to drinking water and sanitation facilities. Methods: We used a Bayesian geostatistical model and data from 600 sources across more than 88 low-income and middle-income countries (LMICs) to estimate access to drinking water and sanitation facilities on continuous continent-wide surfaces from 2000 to 2017, and aggregated results to policy-relevant administrative units. We estimated mutually exclusive and collectively exhaustive subcategories of facilities for drinking water (piped water on or off premises, other improved facilities, unimproved, and surface water) and sanitation facilities (septic or sewer sanitation, other improved, unimproved, and open defecation) with use of ordinal regression. We also estimated the number of diarrhoeal deaths in children younger than 5 years attributed to unsafe facilities and estimated deaths that were averted by increased access to safe facilities in 2017, and analysed geographical inequality in access within LMICs. Findings: Across LMICs, access to both piped water and improved water overall increased between 2000 and 2017, with progress varying spatially. For piped water, the safest water facility type, access increased from 40·0% (95% uncertainty interval [UI] 39·4–40·7) to 50·3% (50·0–50·5), but was lowest in sub-Saharan Africa, where access to piped water was mostly concentrated in urban centres. Access to both sewer or septic sanitation and improved sanitation overall also increased across all LMICs during the study period. For sewer or septic sanitation, access was 46·3% (95% UI 46·1–46·5) in 2017, compared with 28·7% (28·5–29·0) in 2000. Although some units improved access to the safest drinking water or sanitation facilities since 2000, a large absolute number of people continued to not have access in several units with high access to such facilities (>80%) in 2017. More than 253 000 people did not have access to sewer or septic sanitation facilities in the city of Harare, Zimbabwe, despite 88·6% (95% UI 87·2–89·7) access overall. Many units were able to transition from the least safe facilities in 2000 to safe facilities by 2017; for units in which populations primarily practised open defecation in 2000, 686 (95% UI 664–711) of the 1830 (1797–1863) units transitioned to the use of improved sanitation. Geographical disparities in access to improved water across units decreased in 76·1% (95% UI 71·6–80·7) of countries from 2000 to 2017, and in 53·9% (50·6–59·6) of countries for access to improved sanitation, but remained evident subnationally in most countries in 2017. Interpretation: Our estimates, combined with geospatial trends in diarrhoeal burden, identify where efforts to increase access to safe drinking water and sanitation facilities are most needed. By highlighting areas with successful approaches or in need of targeted interventions, our estimates can enable precision public health to effectively progress towards universal access to safe water and sanitation

Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980-2015 : a systematic analysis for the Global Burden of Disease Study 2015

Background Improving survival and extending the longevity of life for all populations requires timely, robust evidence on local mortality levels and trends. The Global Burden of Disease 2015 Study (GBD 2015) provides a comprehensive assessment of all-cause and cause-specific mortality for 249 causes in 195 countries and territories from 1980 to 2015. These results informed an in-depth investigation of observed and expected mortality patterns based on sociodemographic measures. Methods We estimated all-cause mortality by age, sex, geography, and year using an improved analytical approach originally developed for GBD 2013 and GBD 2010. Improvements included refinements to the estimation of child and adult mortality and corresponding uncertainty, parameter selection for under-5 mortality synthesis by spatiotemporal Gaussian process regression, and sibling history data processing. We also expanded the database of vital registration, survey, and census data to 14 294 geography-year datapoints. For GBD 2015, eight causes, including Ebola virus disease, were added to the previous GBD cause list for mortality. We used six modelling approaches to assess cause-specific mortality, with the Cause of Death Ensemble Model (CODEm) generating estimates for most causes. We used a series of novel analyses to systematically quantify the drivers of trends in mortality across geographies. First, we assessed observed and expected levels and trends of cause-specific mortality as they relate to the Socio-demographic Index (SDI), a summary indicator derived from measures of income per capita, educational attainment, and fertility. Second, we examined factors affecting total mortality patterns through a series of counterfactual scenarios, testing the magnitude by which population growth, population age structures, and epidemiological changes contributed to shifts in mortality. Finally, we attributed changes in life expectancy to changes in cause of death. We documented each step of the GBD 2015 estimation processes, as well as data sources, in accordance with Guidelines for Accurate and Transparent Health Estimates Reporting (GATHER). Findings Globally, life expectancy from birth increased from 61.7 years (95% uncertainty interval 61.4-61.9) in 1980 to 71.8 years (71.5-72.2) in 2015. Several countries in sub-Saharan Africa had very large gains in life expectancy from 2005 to 2015, rebounding from an era of exceedingly high loss of life due to HIV/AIDS. At the same time, many geographies saw life expectancy stagnate or decline, particularly for men and in countries with rising mortality from war or interpersonal violence. From 2005 to 2015, male life expectancy in Syria dropped by 11.3 years (3.7-17.4), to 62.6 years (56.5-70.2). Total deaths increased by 4.1% (2.6-5.6) from 2005 to 2015, rising to 55.8 million (54.9 million to 56.6 million) in 2015, but age-standardised death rates fell by 17.0% (15.8-18.1) during this time, underscoring changes in population growth and shifts in global age structures. The result was similar for non-communicable diseases (NCDs), with total deaths from these causes increasing by 14.1% (12.6-16.0) to 39.8 million (39.2 million to 40.5 million) in 2015, whereas age-standardised rates decreased by 13.1% (11.9-14.3). Globally, this mortality pattern emerged for several NCDs, including several types of cancer, ischaemic heart disease, cirrhosis, and Alzheimer's disease and other dementias. By contrast, both total deaths and age-standardised death rates due to communicable, maternal, neonatal, and nutritional conditions significantly declined from 2005 to 2015, gains largely attributable to decreases in mortality rates due to HIV/AIDS (42.1%, 39.1-44.6), malaria (43.1%, 34.7-51.8), neonatal preterm birth complications (29.8%, 24.8-34.9), and maternal disorders (29.1%, 19.3-37.1). Progress was slower for several causes, such as lower respiratory infections and nutritional deficiencies, whereas deaths increased for others, including dengue and drug use disorders. Age-standardised death rates due to injuries significantly declined from 2005 to 2015, yet interpersonal violence and war claimed increasingly more lives in some regions, particularly in the Middle East. In 2015, rotaviral enteritis (rotavirus) was the leading cause of under-5 deaths due to diarrhoea (146 000 deaths, 118 000-183 000) and pneumococcal pneumonia was the leading cause of under-5 deaths due to lower respiratory infections (393 000 deaths, 228 000-532 000), although pathogen-specific mortality varied by region. Globally, the effects of population growth, ageing, and changes in age-standardised death rates substantially differed by cause. Our analyses on the expected associations between cause-specific mortality and SDI show the regular shifts in cause of death composition and population age structure with rising SDI. Country patterns of premature mortality (measured as years of life lost [YLLs]) and how they differ from the level expected on the basis of SDI alone revealed distinct but highly heterogeneous patterns by region and country or territory. Ischaemic heart disease, stroke, and diabetes were among the leading causes of YLLs in most regions, but in many cases, intraregional results sharply diverged for ratios of observed and expected YLLs based on SDI. Communicable, maternal, neonatal, and nutritional diseases caused the most YLLs throughout sub-Saharan Africa, with observed YLLs far exceeding expected YLLs for countries in which malaria or HIV/AIDS remained the leading causes of early death. Interpretation At the global scale, age-specific mortality has steadily improved over the past 35 years; this pattern of general progress continued in the past decade. Progress has been faster in most countries than expected on the basis of development measured by the SDI. Against this background of progress, some countries have seen falls in life expectancy, and age-standardised death rates for some causes are increasing. Despite progress in reducing age-standardised death rates, population growth and ageing mean that the number of deaths from most non-communicable causes are increasing in most countries, putting increased demands on health systems. Copyright (C) The Author(s). Published by Elsevier Ltd.Peer reviewe

Sex Reversal in Zebrafish fancl Mutants is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA-repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination

Increased germ cell apoptosis in <i>fancl</i> mutants at 25 dpf.

<p>Immunodetection of apoptosis by anti-active Caspase-3 in paraffin sections of gonads of wild-type sibling controls (WT) and <i>fancl</i> homozygous mutants (<i>fancl-/-</i>) at 25 dpf (A,B). Presence of Caspase-3-positive cells (shown in red) was lower in gonads of WT (A) than in <i>fancl</i> mutants (B). Gonads outlined by a dashed line (A,B). Bar graph representing the average number of Caspase-3-positive germ cells in each genotype: wild-type sibling controls (WT; n = 6) and <i>fancl</i> homozygous mutants (<i>fancl-/-</i>; n = 6) at 25 dpf (C). Results showed that the average number of apoptotic germ cells in <i>fancl</i> mutants (x– = 99±43) was about three fold higher than in wild-type sibling controls (x– = 35±14), revealing an abnormal increase of germ cell apoptosis in <i>fancl</i> mutants at 25 dpf, a critical period for sex determination (C).</p

A model for zebrafish sex determination: oocyte survival regulated by Tp53-mediated apoptosis can alter gonad fate.

<p>This model suggests germ cell apoptosis as a central feature that can integrate genetic and environmental factors to tip the fate of the gonad towards the female or the male pathway and thus determine zebrafish sex. (A) Zebrafish juveniles initially develop an undifferentiated bipotential immature ovary regardless of their eventual definitive sex. The juvenile gonad contains developing oocytes (shown in yellow), as well as somatic cells that express female-specific markers like <i>cyp19a1a</i> (purple) and early male-specific markers like <i>amh</i> (green). This model suggests that different levels of germ cell apoptosis (indicated as a red gradient box from low (white) to high apoptosis (red)) has the potential to tip the fate of the gonad: high apoptosis (e.g <i>fancl^−/−</i> mutants) tips fate towards the male pathway, while low apoptosis (e.g. <i>fancl^−/− tp53^−/−</i> mutants) tips fate towards the female pathway and rescues the sex-reversal phenotype of <i>fancl</i> mutants. In this model, wild-type zebrafish can enter the male pathway at different times during the fate decision time-window (dashed arrows in apoptosis box) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001034#pgen.1001034-Wang1" target="_blank">[30]</a> and this work), which is probably related to the level of apoptosis that affects oocyte survival in each particular individual. (B) Analysis of somatic markers reported here shows that the survival of oocytes during the fate decision time-window is crucial to maintain and increase expression of <i>cyp19a1a</i> in the somatic cells of the gonad (B, purple gradient) and to down-regulate the expression of <i>amh</i> in the somatic cells of the gonad (B, green gradient), perhaps due to an oocyte-derived signal, which in <i>fancl</i> mutants would be compromised. (C) This gene expression profile feminizes the gonad, oocytes continue to develop, the gonad differentiates as an ovary, and the individual becomes a female. (D) In the absence of oocytes during sex fate decision time, as in <i>fancl</i> mutants, gonads do not maintain <i>cyp19a1a</i> (D, purple gradient), but instead up-regulate <i>amh</i> expression (D, green gradient). (E) This gene expression profile masculinizes the gonad, which differentiates as a mature testis and the individual becomes a male. (F,G) The absence of surviving oocytes in <i>fancl</i>^−/− mutants is probably due to high levels of germ cell apoptosis, which causes all animals to develop as males due to female-to-male sex reversal. This sex reversal phenotype can be rescued by decreasing germ cell apoptosis in double homozygous <i>fancl</i>^−/−;<i>tp53</i>^−/− mutants. Therefore, our analysis of <i>fancl</i> mutants provides evidence supporting the model that the survival of developing oocytes through meiosis, and not the mere presence of germ cells, is a critical factor that tips the fate of the gonad towards the female pathway in zebrafish. (H) Other work has shown that environmental factors such as high temperature can also induce oocyte apoptosis and tip the fate of the gonads towards the male pathway <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001034#pgen.1001034-Uchida2" target="_blank">[71]</a>. In light of this analysis, our model suggests that sex-determining mechanisms in zebrafish integrate signals from genetic and environmental factors that can modify the levels of Tp53-mediated germ cell apoptosis, which influence oocyte survival during the period of gonad fate decision, and tip the fate of the gonad towards the female or the male pathway, thus determining the sex of zebrafish.</p

The Tol2 insertion <i>HG10A</i> disrupts <i>fancl</i> transcripts.

<p>(A) Zebrafish <i>fancl</i> gene structure showing the Tol2 insertion in exon 12 and the position of primer pairs used for RT-PCR experiments (arrows, F1-R1; F2-R2). Numbered boxes represent exons and dashed boxes indicate untranslated regions. (B) Schematic representation from exon 11 to 13 of the wild-type <i>fancl</i> transcript (1.WT) and <i>fancl</i> mutant transcripts (2.<i>fancl</i>Tol2 and 3.<i>fancl</i>D<i>Tol2</i>). The PHD finger domain is highlighted in grey. The Tol2 insertion is shown in black and an arrowhead points to its insertion site in the amino acid sequence in B.2. Predicted protein sequences are shown; the highly conserved Cys and His residues are underlined and the critical Trp is double underlined. Asterisks represent premature stop codons. (C) RT-PCR using as template cDNA of adult testes shows that the 232 bp band containing the intact PHD domain in wild types (amplified by F2-R2 primers) is absent from <i>fancl</i> mutants. The smaller band (174 bp) amplified in <i>fancl</i> mutants corresponds to the <i>fancl</i>D<i>Tol2</i> transcript in B.3. Abbreviations: M, DNA-Marker.</p

Zebrafish germ cells express <i>fancl</i> during gonad development.

<p><i>In situ</i> hybridizations with <i>fancl</i> probe were performed on cryo-sections of wild-type animals at different stages of gonad development. Weak <i>fancl</i> expression signal (arrows) was detected in undifferentiated gonads at 17 days post-fertilization (dpf) (A) and 23 dpf (B). Signal became stronger in germ cells (arrows) of transitioning and immature ovaries (ooplasm of oocytes, arrows in C,E,G) and transitioning and immature testes (D,F,H) at 26, 33, and 37 dpf. In adults, <i>fancl</i> expression was restricted to germ cells, but signal intensity depended on the stage of germ cell differentiation. In adult ovaries (I), early stage IB oocytes (<i>e</i>IB) already showed low <i>fancl</i> expression and late stage IB oocytes (<i>l</i>IB) showed strong <i>fancl</i> signal in the ooplasm, suggesting that <i>fancl</i> expression initiated in early stage IB oocytes. As oogenesis progressed, ooplasm volume increased, cortical alveoli appeared (stage II), yolk accumulated (stage III), and <i>fancl</i> expression signal became diluted. In adult testes (J), <i>fancl</i> expression signal was detected in a subset of cells with large nuclei and morphology consistent with primary spermatocytes (sc), but signal was not detected in cells with small nuclei in an advanced stage of spermatogenesis (i.e. spermatids and sperm (sp)). Oocyte staging is according to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001034#pgen.1001034-Selman1" target="_blank">[49]</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001034#pgen.1001034-RodriguezMari1" target="_blank">[29]</a>. Scale bar: 0.1 mm.</p

The absence of females in <i>fancl</i> homozygous mutants is due to sex reversal.

<p>The bar graph represents percentages of expected (ex, grey bars) and observed (ob, black bars) females and males among 211 progeny from a cross of <i>fancl</i> heterozygous females (<i>fancl</i>^+/−) to <i>fancl</i> homozygous mutant males (<i>fancl</i>^−/−). Total numbers (n) and percentages (%) of animals in each category are indicated on the graph. The expected ratio of female heterozygotes to male heterozygotes to female homozygous mutants to male homozygous mutants is 1∶1∶1∶1, but we observed a ratio of about 1∶1∶0∶2 (46 <i>fancl</i>^+/− females: 62 <i>fancl</i>^+/− males: 0 <i>fancl</i>^−/− females: 103 <i>fancl</i>^−/− males). This result rules out the hypothesis that homozygous mutant females die, but is predicted by the hypothesis that homozygous mutants that otherwise would have become females develop instead as males.</p