Non-canonical Wnt pathway expression in the developing mouse and human retina.

The non-canonical Wnt pathway is an evolutionarily conserved pathway essential for tissue patterning and development across species and tissues. In mammals, this pathway plays a role in neuronal migration, dendritogenesis, axon growth, and synapse formation. However, its role in development and synaptogenesis of the human retina remains less established. In order to address this knowledge gap, we analyzed publicly available single-cell RNA sequencing (scRNAseq) datasets for mouse retina, human retina, and human retinal organoids over multiple developmental time points during outer retinal maturation. We identified ligands, receptors, and mediator genes with a putative role in retinal development, including those with novel or species-specific expression, and validated this expression using fluorescence in situ hybridization (FISH). By quantifying outer nuclear layer (ONL) versus inner nuclear layer (INL) expression, we provide evidence for the differential expression of certain non-canonical Wnt signaling components in the developing mouse and human retina during outer plexiform layer (OPL) development. Importantly, we identified distinct expression patterns of mouse and human FZD3 and WNT10A, as well as previously undescribed expression, such as for mouse Wnt2b in Chat+ starburst amacrine cells. Human retinal organoids largely recapitulated the human non-canonical Wnt pathway expression. Together, this work provides the basis for further study of non-canonical Wnt signaling in mouse and human retinal development and synaptogenesis.

Prenatal air pollution exposure is associated with inflammatory, cardiovascular, and metabolic biomarkers in mothers and newborns.

BACKGROUND: Prenatal air pollution exposure has been associated with individual inflammatory, cardiovascular, and metabolic biomarkers in mothers and neonates. However, studies of air pollution and a comprehensive panel of biomarkers across maternal and cord blood samples remain limited. Few studies used data-driven methods to identify biomarker groupings that converge biomarkers from multiple biological pathways. This study aims to investigate the impacts of prenatal air pollution on groups of biomarkers in maternal and cord blood samples. METHODS: In the Maternal And Developmental Risks from Environmental and Social Stressors (MADRES) cohort, 87 biomarkers were quantified from 45 trimester 1 maternal blood and 55 cord blood samples. Pregnancy and trimester 1-averaged concentrations of particulate matter ≤2.5 µm and ≤10 µm in diameter (PM2.5 and PM10), nitrogen dioxide (NO2), and ozone (O3) were estimated, using inverse distance squared weighted spatial interpolation from regulatory air monitoring stations. Traffic-related NOx was assessed using California Line Source Dispersion Model: freeway/highway roads, non-freeway major roads, non-freeway minor roads, and their sum as total NOx. Elastic Net (EN) regression within the rexposome R package was used to group biomarkers and assess their associations with air pollution. RESULTS: In maternal samples, trimester 1-averaged PM10 was associated with elevated inflammation biomarkers and lowered cardiovascular biomarkers. NO2 exhibited positive associations with cardiovascular and inflammation markers. O3 was inversely associated with inflammation, metabolic, and cardiovascular biomarkers. In cord blood, pregnancy-averaged PM2.5 was associated with higher cardiovascular biomarkers and lower metabolic biomarkers. PM10 was associated with lower inflammation and higher cardiovascular biomarkers. Total and major road NOx was associated with lower cardiovascular biomarkers. CONCLUSION: Prenatal air pollution exposure was associated with changes in biomarkers related to inflammation, cardiovascular, metabolic, cancer, and neurological function in both mothers and neonates. This study shed light on mechanisms by which air pollution can influence biological function during pregnancy.

Organophosphate ester flame retardant chemicals and maternal depression during pregnancy.

BACKGROUND: Depression substantially contributes to pregnancy-related morbidity, and pregnancy is increasingly recognized as a vulnerable window for exposure effects on maternal mental health. Exposures to organophosphate esters (OPEs) are ubiquitous and may have neurotoxic effects; however, their impacts on prenatal depression remain unknown. We evaluated associations of third trimester OPE metabolites on maternal depressive symptoms during pregnancy. METHODS: This study included 422 participants in the Maternal and Developmental Risks from Environmental and Social Stressors (MADRES) cohort, a prospective pregnancy cohort of primarily low-income and Hispanic participants residing in Los Angeles, California. We measured concentrations of nine OPEs in third trimester spot urine samples (mean gestational age = 31.5 ± 2.0 weeks). Using the Center for Epidemiologic Studies-Depression (CES-D) scale, we classified participants as having probable depression during pregnancy (N = 137) or not (N = 285) if one or more CES-D scores administered at each trimester met the suggested cutoff score for clinically significant depressive symptoms (≥16). We estimated associations of prenatal OPE metabolite concentrations in tertiles and risk of prenatal depression using modified Log-Poisson regression. We examined associations of the OPE mixture on depression during pregnancy using Bayesian kernel machine regression (BKMR). RESULTS: Participants with the highest tertiles of DPHP and BDCIPP exposure had a 67% (95% CI: 22%, 128%) and 47% (95% CI: 4%, 108%) increased risk of maternal depressive symptoms during pregnancy, respectively. No associations between other OPE metabolites and maternal depression symptoms were observed. In mixture analyses, we observed a positive and linear association between higher exposure to the OPE metabolite mixture and odds of prenatal maternal depression, primarily driven by DPHP. CONCLUSIONS: Our findings provide new evidence of associations between frequently detected OPE metabolites on maternal depression symptoms during pregnancy. Results could inform future intervention efforts aimed at reducing perinatal maternal depression.

Stepwise developmental mimicry generates proximal-biased kidney organoids.

The kidney maintains body fluid homeostasis by reabsorbing essential compounds and excreting waste. Proximal tubule cells, crucial for renal reabsorption of a range of sugars, ions, and amino acids, are highly susceptible to damage, leading to pathologies necessitating dialysis and kidney transplants. While human pluripotent stem cell-derived kidney organoids are used for modeling renal development, disease, and injury, the formation of proximal nephron cells in these 3D structures is incomplete. Here, we describe how to drive the development of proximal tubule precursors in kidney organoids by following a blueprint of in vivo human nephrogenesis. Transient manipulation of the PI3K signaling pathway activates Notch signaling in the early nephron and drives nephrons toward a proximal precursor state. These "proximal-biased" (PB) organoid nephrons proceed to generate proximal nephron precursor cells. Single-cell transcriptional analyses across the organoid nephron differentiation, comparing control and PB types, confirm the requirement of transient Notch signaling for proximal development. Indicative of functional maturity, PB organoids demonstrate dextran and albumin uptake, akin to in vivo proximal tubules. Moreover, PB organoids are highly sensitive to nephrotoxic agents, display an injury response, and drive expression of HAVCR1 / KIM1 , an early proximal-specific marker of kidney injury. Injured PB organoids show evidence of collapsed tubules, DNA damage, and upregulate the injury-response marker SOX9 . The PB organoid model therefore has functional relevance and potential for modeling mechanisms underpinning nephron injury. These advances improve the use of iPSC-derived kidney organoids as tools to understand developmental nephrology, model disease, test novel therapeutics, and for understanding human renal physiology.

Maternal Dietary Patterns During Pregnancy Are Linked to Hypertensive Disorders of Pregnancy Among a Predominantly Low-Income US Hispanic/Latina Pregnancy Cohort.

BACKGROUND: Diet during pregnancy may be a potential intervention for preventing hypertensive disorders of pregnancy that disproportionally burdens Hispanic/Latina women. METHODS AND RESULTS: The MADRES (Maternal And Developmental Risks from Environmental and Social stressors) study (n=451) is a prospective pregnancy cohort of predominantly low-income Hispanic/Latina women in Los Angeles, California, who completed up to 2 staff-administered 24-hour dietary recalls in the third trimester of pregnancy. Hypertensive disorders of pregnancy were abstracted from medical records and based on a physician's diagnosis or systolic or diastolic blood pressure (≥140 or ≥90 mm Hg, respectively) at ≥2 consecutive prenatal visits. Using multivariable logistic regression, we evaluated associations of 2 previously derived dietary patterns in this population (solid fats, refined grains, and cheese and vegetables, oils, and fruit) and the Healthy Eating Index 2015 with (1) gestational hypertension, (2) preeclampsia, and (3) any hypertensive disorder of pregnancy (either gestational hypertension or preeclampsia). In separate models, we additionally tested interactions with prepregnancy body mass index. Comparing highest-to-lowest quartiles, the solid fats, refined grains, and cheese dietary pattern was associated with an increased odds of any hypertensive disorder of pregnancy (odds ratio [OR], 3.99 [95% CI, 1.44-11.0]; Ptrend=0.014) and preeclampsia (OR, 4.10 [95% CI, 1.25-13.5]; Ptrend=0.036), whereas the vegetables, oils, and fruit pattern was associated with reduced odds of preeclampsia (OR, 0.32 [95% CI, 0.10-0.99]; Ptrend=0.041). Among the overweight prepregnancy body mass index category, inverse associations of vegetables, oils, and fruit and Healthy Eating Index 2015 with preeclampsia were more pronounced (both Pinteractions=0.017). Healthy Eating Index 2015 findings were generally nonsignificant. CONCLUSIONS: While the solid fats, refined grains, and cheese diet was strongly associated with preeclampsia during pregnancy, findings suggest the vegetables, oils, and fruit diet may be more relevant than Healthy Eating Index 2015 for preventing preeclampsia among low-income Hispanic/Latina women.

Defective mesenchymal Bmpr1a-mediated BMP signaling causes congenital pulmonary cysts.

Abnormal lung development can cause congenital pulmonary cysts, the mechanisms of which remain largely unknown. Although the cystic lesions are believed to result directly from disrupted airway epithelial cell growth, the extent to which developmental defects in lung mesenchymal cells contribute to abnormal airway epithelial cell growth and subsequent cystic lesions has not been thoroughly examined. In the present study using genetic mouse models, we dissected the roles of bone morphogenetic protein (BMP) receptor 1a (Bmpr1a)-mediated BMP signaling in lung mesenchyme during prenatal lung development and discovered that abrogation of mesenchymal Bmpr1a disrupted normal lung branching morphogenesis, leading to the formation of prenatal pulmonary cystic lesions. Severe deficiency of airway smooth muscle cells and subepithelial elastin fibers were found in the cystic airways of the mesenchymal Bmpr1a knockout lungs. In addition, ectopic mesenchymal expression of BMP ligands and airway epithelial perturbation of the Sox2-Sox9 proximal-distal axis were detected in the mesenchymal Bmpr1a knockout lungs. However, deletion of Smad1/5, two major BMP signaling downstream effectors, from the lung mesenchyme did not phenocopy the cystic abnormalities observed in the mesenchymal Bmpr1a knockout lungs, suggesting that a Smad-independent mechanism contributes to prenatal pulmonary cystic lesions. These findings reveal for the first time the role of mesenchymal BMP signaling in lung development and a potential pathogenic mechanism underlying congenital pulmonary cysts.

Congenital disorders are medical conditions that are present from birth. Although many congenital disorders are rare, they can have a severe impact on the quality of life of those affected. For example, congenital pulmonary airway malformation (CPAM) is a rare congenital disorder that occurs in around 1 out of every 25,000 pregnancies. In CPAM, abnormal, fluid-filled sac-like pockets of tissue, known as cysts, form within the lungs of unborn babies. After birth, these cysts become air-filled and do not behave like normal lung tissue and stop a baby's lungs from working properly. In severe cases, babies with CPAM need surgery immediately after birth. We still do not understand exactly what the underlying causes of CPAM might be. CPAM is not considered to be hereditary ­ that is, it does not appear to be passed down in families ­ nor is it obviously linked to any environmental factors. CPAM is also very difficult to study, because researchers cannot access tissue samples during the critical early stages of the disease. To overcome these difficulties, Luo et al. wanted to find a way to study CPAM in the laboratory. First, they developed a non-human animal 'model' that naturally forms CPAM-like lung cysts, using genetically modified mice where the gene for the signaling molecule Bmpr1a had been deleted in lung cells. Normally, Bmpr1a is part of a set of the molecular instructions, collectively termed BMP signaling, which guide healthy lung development early in life. However, mouse embryos lacking Bmpr1a developed abnormal lung cysts that were similar to those found in CPAM patients, suggesting that problems with BMP signalling might also trigger CPAM in humans. Luo et al. also identified several other genes in the Bmpr1a-deficient mouse lungs that had abnormal patterns of activity. All these genes were known to be controlled by BMP signaling, and to play a role in the development and organisation of lung tissue. This suggests that when these genes are not controlled properly, they could drive formation of CPAM cysts when BMP signaling is compromised. This work is a significant advance in the tools available to study CPAM. Luo et al.'s results also shed new light on the molecular mechanisms underpinning this rare disorder. In the future, Luo et al. hope this knowledge will help us develop better treatments for CPAM, or even help to prevent it altogether.

Joint effects of traffic-related air pollution and hypertensive disorders of pregnancy on maternal postpartum depressive and anxiety symptoms.

BACKGROUND: Ambient air pollution has been linked to postpartum depression. However, few studies have investigated the effects of traffic-related NOx on postpartum depression and whether any pregnancy-related factors might increase susceptibility. OBJECTIVES: To evaluate the association between traffic-related NOx and postpartum depressive and anxiety symptoms, and effect modification by pregnancy-related hypertension. METHODS: This study included 453 predominantly low-income Hispanic/Latina women in the MADRES cohort. Daily traffic-related NOx concentrations by road class were estimated using the California LINE-source dispersion model (CALINE4) at participants' residential locations and averaged across pregnancy. Postpartum depressive and anxiety symptoms were evaluated by a validated questionnaire (Postpartum Distress Measure, PDM) at 1, 3, 6 and 12 months postpartum. Multivariate linear regressions were performed to estimate the associations at each timepoint. Interaction terms were added to the linear models to assess effect modification by hypertensive disorders of pregnancy (HDPs). Repeated measurement analyses were conducted by using mixed effect models. RESULTS: We found prenatal traffic-related NOx was associated with increased PDM scores. Specifically, mothers exposed to an IQR (0.22 ppb) increase in NOx from major roads had 3.78% (95% CI: 0.53-7.14%) and 5.27% (95% CI: 0.33-10.45%) significantly higher 3-month and 12-month PDM scores, respectively. Similarly, in repeated measurement analyses, higher NOx from major roads was associated with 3.06% (95% CI: 0.43-5.76%) significantly higher PDM scores across the first year postpartum. Effect modification by HDPs was observed: higher freeway/highway and total NOx among mothers with HDPs were associated with significantly higher PDM scores at 12 months postpartum compared to those without HDPs. IMPACT: This study shows that prenatal traffic-related air pollution was associated with postpartum depressive and anxiety symptoms. The study also found novel evidence of greater susceptibility among women with HDPs, which advances the understanding of the relationships between air pollution, maternal cardiometabolic health during pregnancy and postpartum mental health. Our study has potential implications for clinical intervention to mitigate the effects of traffic-related pollution on postpartum mental health disorders. The findings can also offer valuable insights into urban planning strategies concerning the implementation of emission control measures and the creation of green spaces.

Increased Risk of Gestational Hypertension by Periconceptional Exposure to Ambient Air Pollution and Effect Modification by Prenatal Depression.

BACKGROUND: Air pollution has been associated with gestational hypertension (GH) and preeclampsia, but susceptible windows of exposure and potential vulnerability by comorbidities, such as prenatal depression, remain unclear. METHODS: We ascertained GH and preeclampsia cases in a prospective pregnancy cohort in Los Angeles, CA. Daily levels of ambient particulate matters (with a diameter of ≤10 µm [PM10] or ≤2.5 µm [PM2.5]), nitrogen dioxide, and ozone were averaged for each week from 12 weeks preconception to 20 gestational weeks. We used distributed lag models to identify susceptible exposure windows, adjusting for potential confounders. Analyses were additionally stratified by probable prenatal depression to explore population vulnerability. RESULTS: Among 619 participants, 60 developed preeclampsia and 42 developed GH. We identified a susceptible window for exposure to PM2.5 from 1 week preconception to 11 weeks postconception: higher exposure (5 µg/m3) within this window was associated with an average of 8% (95% CI, 1%-15%) higher risk of GH. Among participants with probable prenatal depression (n=179; 32%), overlapping sensitive windows were observed for all pollutants from 8 weeks before to 10 weeks postconception with increased risk of GH (PM2.5, 16% [95% CI, 3%-31%]; PM10, 39% [95% CI, 13%-72%]; nitrogen dioxide, 65% [95% CI, 17%-134%]; and ozone, 45% [95% CI, 9%-93%]), while the associations were close to null among those without prenatal depression. Air pollutants were not associated with preeclampsia in any analyses. CONCLUSIONS: We identified periconception through early pregnancy as a susceptible window of air pollution exposure with an increased risk of GH. Prenatal depression increases vulnerability to air pollution exposure and GH.

Sources of personal PM2.5 exposure during pregnancy in the MADRES cohort.

BACKGROUND: Personal exposure to fine particulate matter (PM2.5) is impacted by different sources each with different chemical composition. Determining these sources is important for reducing personal exposure and its health risks especially during pregnancy. OBJECTIVE: Identify main sources and their contributions to the personal PM2.5 exposure in 213 women in the 3rd trimester of pregnancy in Los Angeles, CA. METHODS: We measured 48-hr integrated personal PM2.5 exposure and analyzed filters for PM2.5 mass, elemental composition, and optical carbon fractions. We used the EPA Positive Matrix Factorization (PMF) model to resolve and quantify the major sources of personal PM2.5 exposure. We then investigated bivariate relationships between sources, time-activity patterns, and environmental exposures in activity spaces and residential neighborhoods to further understand sources. RESULTS: Mean personal PM2.5 mass concentration was 22.3 (SD = 16.6) µg/m3. Twenty-five species and PM2.5 mass were used in PMF with a final R2 of 0.48. We identified six sources (with major species in profiles and % contribution to PM2.5 mass) as follows: secondhand smoking (SHS) (brown carbon, environmental tobacco smoke; 65.3%), fuel oil (nickel, vanadium; 11.7%), crustal (aluminum, calcium, silicon; 11.5%), fresh sea salt (sodium, chlorine; 4.7%), aged sea salt (sodium, magnesium, sulfur; 4.3%), and traffic (black carbon, zinc; 2.6%). SHS was significantly greater in apartments compared to houses. Crustal source was correlated with more occupants in the household. Aged sea salt increased with temperature and outdoor ozone, while fresh sea salt was highest on days with westerly winds from the Pacific Ocean. Traffic was positively correlated with ambient NO2 and traffic-related NOx at residence. Overall, 76.8% of personal PM2.5 mass came from indoor or personal compared to outdoor sources. IMPACT: We conducted source apportionment of personal PM2.5 samples in pregnancy in Los Angeles, CA. Among identified sources, secondhand smoking contributed the most to the personal exposure. In addition, traffic, crustal, fuel oil, fresh and aged sea salt sources were also identified as main sources. Traffic sources contained markers of combustion and non-exhaust wear emissions. Crustal source was correlated with more occupants in the household. Aged sea salt source increased with temperature and outdoor ozone and fresh sea salt source was highest on days with westerly winds from the Pacific Ocean.

Single cell transcriptomics reveals early photoreceptor states, cell-specific transcript isoforms, and cancer-predisposing features.

Human cone photoreceptors differ from rods and serve as the retinoblastoma cell-of-origin. Here, we used deep full-length single-cell RNA-sequencing to distinguish post-mitotic cone and rod developmental states and cone-specific features that contribute to retinoblastomagenesis. The analyses revealed early post-mitotic cone- and rod-directed populations characterized by higher THRB or NRL regulon activities, an immature photoreceptor precursor population with concurrent cone and rod gene and regulon expression, and distinct early and late cone and rod maturation states distinguished by maturation-associated declines in RAX regulon activity. Unexpectedly, both L/M cone and rod precursors co-expressed NRL and THRB RNAs, yet they differentially expressed functionally antagonistic NRL isoforms and prematurely terminated THRB transcripts. Early L/M cone precursors exhibited successive expression of lncRNAs along with MYCN, which composed the seventh most L/M-cone-specific regulon, and SYK, which contributed to the early cone precursors' proliferative response to RB1 loss. These findings reveal previously unrecognized photoreceptor precursor states and a role for early cone-precursor-intrinsic SYK expression in retinoblastoma initiation.

Long-term expandable mouse and human-induced nephron progenitor cells enable kidney organoid maturation and modeling of plasticity and disease.

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration.

Prenatal Perfluorooctanoic Acid (PFOA) Exposure Is Associated With Lower Infant Birthweight Within the MADRES Pregnancy Cohort.

Introduction: Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are persistent synthetic chemicals found in household products that can cross the placenta during pregnancy. We investigated whether PFAS exposure during pregnancy was associated with infant birth outcomes in a predominantly urban Hispanic population. Methods: Serum concentrations of perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) were measured in 342 prenatal biospecimens (mean gestational age: 21 ± 9 weeks) from participants in the ongoing Maternal And Developmental Risks from Environmental and Social Stressors (MADRES) cohort. PFAS compounds were modeled continuously or categorically, depending on the percentage of samples detected. The birth outcomes assessed were birthweight, gestational age at birth, and birthweight for gestational age (BW-for-GA) z-scores that accounted for parity or infant sex. Single pollutant and multipollutant linear regression models were performed to evaluate associations between PFAS exposures and birth outcomes, adjusting for sociodemographic, perinatal, and study design covariates. Results: Maternal participants (n = 342) were on average 29 ± 6 years old at study entry and were predominantly Hispanic (76%). Infants were born at a mean of 39 ± 2 weeks of gestation and weighed on average 3,278 ± 522 g. PFOS and PFHxS were detected in 100% of the samples while PFNA, PFOA, and PFDA were detected in 70%, 65%, and 57% of the samples, respectively. PFAS levels were generally lower in this cohort than in comparable cohorts. Women with detected levels of PFOA during pregnancy had infants weighing on average 119.7 g less (95% CI -216.7, -22.7) than women with undetected levels of PFOA in adjusted single pollutant models. PFOA results were also statistically significant in BW-for-GA z-score models that were specific for sex or parity. In models that were mutually adjusted for five detected PFAS compounds, PFOA results remained comparable; however, the association was only significant in BW-for-GA z-scores that were specific for parity (ß = -0.3; 95% CI -0.6, -0.01). We found no significant adjusted associations with the remaining PFAS concentrations and the birth outcomes assessed. Conclusion: Prenatal exposure to PFOA was associated with lower birthweight in infants, suggesting that exposure to these chemicals during critical periods of development might have important implications for children's health.