Embryonic Day Research Articles

Hypothalamic vasopressin sex differentiation is observed by embryonic day 15 in mice and is disrupted by the xenoestrogen bisphenol A

Arginine vasopressin (AVP) neurons of the hypothalamic paraventricular region (AVPPVN) mediate sex-biased social behaviors across most species, including mammals. In mice, neural sex differences are thought to be established during a critical window around birth ( embryonic (E) day 18 to postnatal (P) day 2) whereby circulating testosterone from the fetal testis is converted to estrogen in sex-dimorphic brain regions. Here, we found that AVPPVN neurons are sexually dimorphic by E15.5, prior to this critical window, and that gestational bisphenol A (BPA) exposure permanently masculinized female AVPPVN neuronal numbers, projections, and electrophysiological properties, causing them to display male-like phenotypes into adulthood. Moreover, we showed that nearly twice as many neurons that became AVP+ by P0 were born at E11 in males and BPA-exposed females compared to control females, suggesting that AVPPVN neuronal masculinization occurs between E11 and P0. We further narrowed this sensitive period to around the timing of neurogenesis by demonstrating that exogenous estrogen exposure from E14.5 to E15.5 masculinized female AVPPVN neuronal numbers, whereas a pan-estrogen receptor antagonist exposed from E13.5 to E15.5 blocked masculinization of males. Finally, we showed that restricting BPA exposure to E7.5-E15.5 caused adult females to display increased social dominance over control females, consistent with an acquisition of male-like behaviors. Our study reveals an E11.5 to E15.5 window of estrogen sensitivity impacting AVPPVN sex differentiation, which is impacted by prenatal BPA exposure.

Hydrogen gas inhalation ameliorates LPS-induced BPD by inhibiting inflammation via regulating the TLR4–NFκB–IL6/NLRP3 signaling pathway in the placenta

IntroductionHydrogen (H2) is regarded as a novel therapeutic agent against several diseases owing to its inherent biosafety. Bronchopulmonary dysplasia (BPD) has been widely considered among adverse pregnancy outcomes, without effective treatment. Placenta plays a role in defense, synthesis, and immunity, which provides a new perspective for the treatment of BPD. This study aimed to investigate if H2 reduced the placental inflammation to protect the neonatal rat against BPD damage and potential mechanisms.MethodsWe induced neonatal BPD model by injecting lipopolysaccharide (LPS, 1 µg) into the amniotic fluid at embryonic day 16.5 as LPS group. LPS + H2 group inhaled 42% H2 gas (4 h/day) until the samples were collected. We primarily analyzed the neonatal outcomes and then compared inflammatory levels from the control group (CON), LPS group and LPS + H2 group. HE staining was performed to evaluate inflammatory levels. RNA sequencing revealed dominant differentially expressed genes. Bioinformatics analysis (GO and KEGG) of RNA-seq was applied to mine the signaling pathways involved in protective effect of H2 on the development of LPS-induced BPD. We further used qRT-PCR, Western blot and ELISA methods to verify differential expression of mRNA and proteins. Moreover, we verified the correlation between the upstream signaling pathways and the downstream targets in LPS-induced BPD model.ResultsUpon administration of H2, the inflammatory infiltration degree of the LPS-induced placenta was reduced, and infiltration significantly narrowed. Hydrogen normalized LPS-induced perturbed lung development and reduced the death ratio of the fetus and neonate. RNA-seq results revealed the importance of inflammatory response biological processes and Toll-like receptor signaling pathway in protective effect of hydrogen on BPD. The over-activated upstream signals [Toll-like receptor 4 (TLR4), nuclear factor kappa-B p65 (NF-κB p65), Caspase1 (Casp1) and NLR family pyrin domain containing 3 (NLRP3) inflammasome] in LPS placenta were attenuated by H2 inhalation. The downstream targets, inflammatory cytokines/chemokines [interleukin (IL)-6, IL-18, IL-1β, C–C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 1 (CXCL1)], were decreased both in mRNA and protein levels by H2 inhalation in LPS-induced placentas to rescue them from BPD. Correlation analysis displayed a positive association of TLR4-mediated signaling pathway both proinflammatory cytokines and chemokines in placenta.ConclusionH2 inhalation ameliorates LPS-induced BPD by inhibiting excessive inflammatory cytokines and chemokines via the TLR4–NFκB–IL6/NLRP3 signaling pathway in placenta and may be a potential therapeutic strategy for BPD.

Bone Regenerative Effect of Injectable Hypoxia Preconditioned Serum-Fibrin (HPS-F) in an Ex Vivo Bone Defect Model.

Biofunctionalized hydrogels are widely used in tissue engineering for bone repair. This study examines the bone regenerative effect of the blood-derived growth factor preparation of Hypoxia Preconditioned Serum (HPS) and its fibrin-hydrogel formulation (HPS-F) on drilled defects in embryonic day 19 chick femurs. Measurements of bone-related growth factors in HPS reveal significant elevations of Osteopontin, Osteoprotegerin, and soluble-RANKL compared with normal serum (NS) but no detection of BMP-2/7 or Osteocalcin. Growth factor releases from HPS-F are measurable for at least 7 days. Culturing drilled femurs organotypically on a liquid/gas interface with HPS media supplementation for 10 days demonstrates a 34.6% increase in bone volume and a 52.02% increase in bone mineral density (BMD) within the defect area, which are significantly higher than NS and a basal-media-control, as determined by microcomputed tomography. HPS-F-injected femur defects implanted on a chorioallantoic membrane (CAM) for 7 days exhibit an increase in bone mass of 123.5% and an increase in BMD of 215.2%, which are significantly higher than normal-serum-fibrin (NS-F) and no treatment. Histology reveals calcification, proteoglycan, and collagen fiber deposition in the defect area of HPS-F-treated femurs. Therefore, HPS-F may offer a promising and accessible therapeutic approach to accelerating bone regeneration by a single injection into the bone defect site.

Transcriptome analysis of long non-coding RNA associated with embryonic muscle development in chickens

ABSTRACT 1. Skeletal muscle is an important component of chicken carcass. In chickens, the number of muscle fibres is fixed during the embryonic period, and muscle development during the embryonic period determines the muscle development potential after hatching. 2. Beijing-You (BY) and Cornish (CN) chickens show completely different growth rates and body types, and two breeds were used in this study to explore the role of lncRNAs in muscle development during different chicken embryonic periods. A systematic analysis of lncRNAs and mRNAs were conducted in the pectoral muscle tissues of BY and CN chickens at embryonic days 11 (ED11), 13 (ED13), 15 (ED15), 17 (ED17), and 1-day-old (D1) using RNA-seq. A total of 4,104 differentially expressed transcripts (DETs) were identified among the five stages, including 2,359 lncRNAs and 1,745 mRNAs. 3. The number of DETs between the two breeds at ED17 (1,658 lncRNAs and 1,016 mRNAs) was much higher than the total number of DET at all the other stages (692 lncRNAs and 729 mRNAs), indicating that the two breeds show the largest difference in gene regulation at ED17. 4. Correlation analysis was performed for all differentially expressed lncRNAs and mRNAs during the five periods. Forty-three, cis interaction pairs of lncRNA-mRNA related to chicken muscle development were predicted. The expression of four pairs was verified, and the results showed MSTRG.12395.2-FGFBP2 and MSTRG.18590.6-FMOD were significantly up-regulated in CN at ED11 compared to BY and might be important candidate genes for embryonic muscle development.

The single-cell chromatin landscape in gonadal cell lineage specification

Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.

Twist2 contributes to skin regeneration and hair follicle formation in mouse fetuses

Unlike adult mammalian wounds, early embryonic mouse skin wounds completely regenerate and heal without scars. Analysis of the underlying molecular mechanism will provide insights into scarless wound healing. Twist2 is an important regulator of hair follicle formation and biological patterning; however, it is unclear whether it plays a role in skin or skin appendage regeneration. Here, we aimed to elucidate Twist2 expression and its role in fetal wound healing. ICR mouse fetuses were surgically wounded on embryonic day 13 (E13), E15, and E17, and Twist2 expression in tissue samples from these fetuses was evaluated via in situ hybridization, immunohistochemistry, and reverse transcription-quantitative polymerase chain reaction. Twist2 expression was upregulated in the dermis of E13 wound margins but downregulated in E15 and E17 wounds. Twist2 knockdown on E13 left visible marks at the wound site, inhibited regeneration, and resulted in defective follicle formation. Twist2-knockdown dermal fibroblasts lacked the ability to undifferentiate. Furthermore, Twist2 hetero knockout mice (Twist + /-) formed visible scars, even on E13, when all skin structures should regenerate. Thus, Twist2 expression correlated with skin texture formation and hair follicle defects in late mouse embryos. These findings may help develop a therapeutic strategy to reduce scarring and promote hair follicle regeneration.

Impact of maternal immune activation and sex on placental and fetal brain cytokine and gene expression profiles in a preclinical model of neurodevelopmental disorders

Maternal inflammation during gestation is associated with a later diagnosis of neurodevelopmental disorders including autism spectrum disorder (ASD). However, the specific impact of maternal immune activation (MIA) on placental and fetal brain development remains insufficiently understood. This study aimed to investigate the effects of MIA by analyzing placental and brain tissues obtained from the offspring of pregnant C57BL/6 dams exposed to polyinosinic: polycytidylic acid (poly I: C) on embryonic day 12.5. Cytokine and mRNA content in the placenta and brain tissues were assessed using multiplex cytokine assays and bulk-RNA sequencing on embryonic day 17.5. In the placenta, male MIA offspring exhibited higher levels of GM-CSF, IL-6, TNFα, and LT-α, but there were no differences in female MIA offspring. Furthermore, differentially expressed genes (DEG) in the placental tissues of MIA offspring were found to be enriched in processes related to synaptic vesicles and neuronal development. Placental mRNA from male and female MIA offspring were both enriched in synaptic and neuronal development terms, whereas females were also enriched for terms related to excitatory and inhibitory signaling. In the fetal brain of MIA offspring, increased levels of IL-28B and IL-25 were observed with male MIA offspring and increased levels of LT-α were observed in the female offspring. Notably, we identified few stable MIA fetal brain DEG, with no male specific difference whereas females had DEG related to immune cytokine signaling. Overall, these findings support the hypothesis that MIA contributes to the sex- specific abnormalities observed in ASD, possibly through altered neuron developed from exposure to inflammatory cytokines. Future research should aim to investigate how interactions between the placenta and fetal brain contribute to altered neuronal development in the context of MIA.

Expression of genes related to ileal barrier function in heritage and modern broiler chickens

ABSTRACT 1. An experiment was conducted to determine differences in the expression of genes encoding intestinal barrier proteins between fast, medium and slow-growing chickens. Chicken breeds Athens Canadian Random Bred (ACRB), Longenecker’s Heritage (LHR), RedBro, Hubbard H1 (HH1), Cobb500 and Ross708 were raised from hatch for 35 d. 2. Ileal samples were collected at embryonic day E19 (−2 days post-hatch), hatch and d 7, 14, 21, 28 and 35 post-hatch to assess the expression of genes encoding tight junction proteins (claudins, CLDN; occludin, OCLN; zonula occludens, ZO; and junctional adhesion molecules, JAM), mucin (Muc2), immunoglobulin A (IgA), polymeric immunoglobulin receptor (pIgR) and fatty acid binding protein (FABP2). 3. Expression of CLDN-1 was increased (p < 0.0001) in LHR compared to Cobb500 while CLDN-5 was increased (p < 0.0001) in ACRB, HH1, RedBro and Ross708 compared to LHR as well as in ACRB compared to Cob500. Occludin was upregulated (p = 0.01) in ACRB and LHR compared to Ross708 at d 14 post-hatch. Expression of ZO-1 was upregulated (p = 0.001) in LHR compared to Ross708, HH1 and Cobb500. Tight junction genes, except CLDN-4, JAM-2 and JAM-3 were downregulated (p < 0.0001) at hatch and d 7 post-hatch. Expression of Muc2 was increased (p < 0.0001) in LHR compared to RedBro and from −2 d to d 7 post-hatch. 4. Immunoglobulin A was increased (p = 0.001) in LHR compared to Ross708 and HH1 at −2 d post-hatch and in LHR compared to ACRB, Cobb500 and Ross708 at hatch. In addition, IgA expression was increased in all breeds at d 14 post-hatch while pIgR was upregulated (p = 0.02) in Cobb500 and Ross708 compared to ACRB, HH1, LHR and RedBro at hatch. 5. The gene expression patterns suggest that selection for growth may have not induced changes in junctional complexes and immune defence genes. However, the results confirmed that the expression of these genes is age dependent.

Effect of in ovo feeding of xylobiose and xylotriose on plasma immunoglobulin, cecal metabolites production, microbial ecology, and metabolic pathways in broiler chickens

BackgroundDietary supplementation of xylooligosaccharides (XOS) has been found to influence gut health by manipulating cecal microbiota and producing microbe-origin metabolites. But no study investigated and compared the effect of in ovo feeding of xylobiose (XOS2) and xylotriose (XOS3) in chickens. This study investigated the effect of in ovo feeding of these XOS compounds on post-hatch gut health parameters in chickens. A total of 144 fertilized chicken eggs were divided into three groups: a) non-injected control (CON), b) XOS2, and c) XOS3. On the 17th embryonic day, the eggs of the XOS2 and XOS3 groups were injected with 3 mg of XOS2 and XOS3 diluted in 0.5 mL of 0.85% normal saline through the amniotic sac. After hatching, the chicks were raised for 21 d. Blood was collected on d 14 to measure plasma immunoglobulin. Cecal digesta were collected for measuring short-chain fatty acids (SCFA) on d 14 and 21, and for microbial ecology and microbial metabolic pathway analyses on d 7 and 21.ResultsThe results were considered significantly different at P < 0.05. ELISA quantified plasma IgA and IgG on d 14 chickens, revealing no differences among the treatments. Gas chromatography results showed no significant differences in the concentrations of cecal SCFAs on d 14 but significant differences on d 21. However, the SCFA concentrations were lower in the XOS3 than in the CON group on d 21. The cecal metagenomics data showed that the abundance of the family Clostridiaceae significantly decreased on d 7, and the abundance of the family Oscillospiraceae increased on d 21 in the XOS2 compared to the CON. There was a reduction in the relative abundance of genus Clostridium sensu stricto 1 in the XOS2 compared to the CON on d 7 and the genus Ruminococcus torques in both XOS2 and XOS3 groups compared to the CON on d 21. The XOS2 and XOS3 groups reduced the genes for chondroitin sulfate degradation I and L-histidine degradation I pathways, which contribute to improved gut health, respectively, in the microbiome on d 7. In contrast, on d 21, the XOS2 and XOS3 groups enriched the thiamin salvage II, L-isoleucine biosynthesis IV, and O-antigen building blocks biosynthesis (E. coli) pathways, which are indicative of improved gut health. Unlike the XOS3 and CON, the microbiome enriched the pathways associated with energy enhancement, including flavin biosynthesis I, sucrose degradation III, and Calvin-Benson-Bassham cycle pathways, in the XOS2 group on d 21.ConclusionIn ovo XOS2 and XOS3 feeding promoted beneficial bacterial growth and reduced harmful bacteria at the family and genus levels. The metagenomic-based microbial metabolic pathway profiling predicted a favorable change in the availability of cecal metabolites in the XOS2 and XOS3 groups. The modulation of microbiota and metabolic pathways suggests that in ovo XOS2 and XOS3 feeding improved gut health during the post-hatch period of broilers.

Ontogeny of hepatic metabolism in two broiler lines divergently selected for the ultimate pH of the Pectoralis major muscle

BackgroundNutrient availability during early stages of development (embryogenesis and the first week post-hatch) can have long-term effects on physiological functions and bird metabolism. The embryo develops in a closed structure and depends entirely on the nutrients and energy available in the egg. The aim of this study was to describe the ontogeny of pathways governing hepatic metabolism that mediates many physiological functions in the pHu + and pHu- chicken lines, which are divergently selected for the ultimate pH of meat, a proxy for muscle glycogen stores, and which differ in the nutrient content and composition of eggs.ResultsWe identified eight clusters of genes showing a common pattern of expression between embryonic day 12 (E12) and day 8 (D8) post-hatch. These clusters were not representative of a specific metabolic pathway or function. On E12 and E14, the majority of genes differentially expressed between the pHu + and pHu- lines were overexpressed in the pHu + line. Conversely, the majority of genes differentially expressed from E18 were overexpressed in the pHu- line. During the metabolic shift at E18, there was a decrease in the expression of genes linked to several metabolic functions (e.g. protein synthesis, autophagy and mitochondrial activity). At hatching (D0), there were two distinct groups of pHu + chicks based on hierarchical clustering; these groups also differed in liver weight and serum parameters (e.g. triglyceride content and creatine kinase activity). At D0 and D8, there was a sex effect for several metabolic pathways. Metabolism appeared to be more active and oriented towards protein synthesis (RPS6) and fatty acid β-oxidation (ACAA2, ACOX1) in males than in females. In comparison, the genes overexpressed in females were related to carbohydrate metabolism (SLC2A1, SLC2A12, FoxO1, PHKA2, PHKB, PRKAB2 and GYS2).ConclusionsOur study provides the first detailed description of the evolution of different hepatic metabolic pathways during the early development of embryos and post-hatching chicks. We found a metabolic orientation for the pHu + line towards proteolysis, glycogen degradation, ATP synthesis and autophagy, likely in response to a higher energy requirement compared with pHu- embryos. The metabolic orientations specific to the pHu + and pHu- lines are established very early, probably in relation with their different genetic background and available nutrients.

β-arrestin1/2 are essential for embryonic lymphatic vessel development

Lymphatic dysfunction can lead to fetal loss due to interstitial edema known as hydrops fetalis during development and lymphedema in adulthood. Lymphedema affects an estimated 250 million individuals worldwide, yet there is no effective treatment available. Numerous studies have established G protein-coupled receptors (GPCRs) as key lymphangiogenic signaling pathways and ideal druggable therapeutical targets. β-arrestins (β-arrestin 1 and 2) are ubiquitously expressed cytosolic adaptor proteins which perform a wide range of functions in GPCR pathways, such as promoting agonist-induced internalization and forming signaling scaffolds with internalized GPCRs in endosomes. However, the roles of β-arrestins in lymphatic vessel development and function remain undefined. We hypothesize that β-arrestin1/2 are essential in embryonic lymphatic vessel development. To test this hypothesis, I used an inducible β-arrestin1fl/fl (Arrb1 fl/fl), β-arrestin2fl/fl (Arrb2 fl/fl);Prox1 CreERT2 mouse line. β-arrestin1 and 2 were deleted by oral gavage of tamoxifen at embryonic day 10.5 (E10.5) and E12.5, critical points for developmental lymphangiogenesis. Compared to Arrb1/2fl/flembryos, the Arrb1/2fl/fl;Prox1CreERT2 embryos exhibit profound hydrops fetalis, subcutaneous hemorrhages, and increased embryonic lethality between E14.5 and E16.5. At E15.5, the Arrb1/2fl/fl;Prox1CreERT2 embryos with edema show decreased weight indicating growth restriction compared to controls. Jugular lymphatic sacs are enlarged and filled with blood in Arrb1/2fl/fl;Prox1CreERT2 embryos compared to controls. Dermal lymphatic vessel length in the Arrb1/2fl/fl;Prox1CreERT2 embryos is shorter than controls. Collectively, our results demonstrate that loss of β-arrestin1/2 expression in lymphatics causes mid-gestational arrest in lymphatic development and fetal growth restriction, indicating that β-arrestins are required for developmental lymphangiogenesis in mammals. American Physiological Society postdoctoral Fellowship (to YT) and NIH R01 HL129086 (to KMC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Perinatal High Fat Diet Exposure Reduces KCC2 Expression and Alters GABAergic Signaling in the Dorsal Motor Nucleus of the Vagus

Perinatal high fat diet (pHFD) exposure alters the development of vagal neurocircuits controlling gastrointestinal (GI) functions resulting in gastric motor dysfunction. GABAergic synaptic inhibition of vagal efferent motoneurons within the dorsal motor nucleus of the vagus (DMV) is critical in the brain’s ability to modulate gastric motility. This inhibition is maintained by KCC2, a Cl− transporter, who determines intracellular chloride concentration. The current study was designed to test the hypothesis that pHFD reduces KCC2 levels in DMV neurons, subsequently decreasing GABAergic inhibition and delaying gastric emptying rates. Pregnant Sprague-Dawley rats were fed either a control or high fat (14% vs 60% kcal from fat, respectively) diet from embryonic day 13, and offspring were used starting postnatal day 28. In vitro electrophysiological recordings were made from adult DMV neurons in brainstem slices. To assess the GABA-mediated inhibition, cell-attached recordings were made to preserve the internal Cl− concentration, and firing rate was quantified before and after application of bicuculline (BIC; 30μM), a GABAA antagonist. To determine the Cl− reversal potential, perforated patch clamp recordings were performed and the response to picospritz application of the GABAA agonist muscimol (100μM) was measured. KCC2 expression was quantified using pharmacology in the in vitro slice preparation, as well as immunofluorescence in brainstem cross-sections. Gastric emptying rates were quantified using the in vivo13C octanoic acid breath test technique. KCC2 transfection and induced expression in the DMV of pHFD rats was achieved using an adeno-associated viral vector. BIC increased firing rate in control DMV neurons (143.1% of baseline), but failed to increase DMV firing rate in DMV neurons from pHFD rats (96.27% of baseline; p=0.0103 using unpaired t-test). When control DMV neurons were treated with VU0240551 (VU; 10μM), a KCC2 blocker, BIC application led to a decrease in the firing rate (3.118 Hz to 1.606 Hz; p=0.0375 via paired t-test), demonstrating that antagonizing KCC2 attenuates GABAergic inhibition at this synapse. In control DMV neurons, the Cl− reversal potential was calculated to be -64.6mV. There was a depolarizing shift in Cl− reversal potential observed in pHFD neurons (-43.0mV; p=0.0007 using unpaired t-test). Application of VU shifted the chloride reversal from -64.6mV to -57.3mV in control neurons (p=0.0416 using a paired t-test). VU application had a reduced impact on pHFD neurons, shifting the reversal from -46.7mV to -45.0mV (p=0.1689 using a paired t-test), suggesting KCC2 downregulation. In fact, compared to control rats, there was a significant reduction in KCC2 expression in the pHFD DMV (2.237% area fluorescence in pHFD vs. 5.335%; p=0.0147 using unpaired t-test). When KCC2 expression was virally induced in young adult pHFD rats, GABA became inhibitory (163.7% of baseline firing rate after BIC application vs. 93.25% in pHFD + empty vector (EV); p=0.0019 using unpaired t-test), the Cl− reversal potential was normalized (-63.0mV vs. -49.7mV in pHFD + EV; p=0.0012 using an unpaired t-test), and gastric emptying rates were accelerated (58.7min vs 72.1min in pHFD + EV). Taken together, these results suggest that the loss of KCC2 function is a driver of the gastric pathophysiology seen in the pHFD rats, and that normalizing KCC2 levels can rescue the cellular and gastric phenotype. DK 111667. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Prenatal AAV9-GFP administration in fetal lambs results in transduction of female germ cells and maternal exposure to virus

Prenatal somatic cell gene therapy (PSCGT) could potentially treat severe, early-onset genetic disorders such as spinal muscular atrophy (SMA) or muscular dystrophy. Given the FDA-approval of adeno-associated virus serotype 9 (AAV9) vectors in infants with SMA, we tested the safety and biodistribution of AAV9-GFP (clinical-grade and dose) in fetal lambs to understand safety and efficacy after umbilical vein (UV) or intracranial (IC) injection on embryonic day 75 (E75). UV-injection led to widespread biodistribution of vector genomes in all examined lamb tissues and in maternal uteruses at harvest (E96 or E140; term=E150). There was robust GFP expression in brain, spinal cord, dorsal root ganglia (DRGs), without DRG toxicity and excellent transduction of diaphragm and quadriceps muscles. However, we found evidence of systemic toxicity (fetal growth restriction) and maternal exposure to the viral vector (transient elevation of total bilirubin and a trend towards elevation in anti-AAV9 antibodies). There were no antibodies against GFP in ewes or lambs. Analysis of fetal gonads demonstrated GFP expression in female (but not male) germ cells, with low levels of integration-specific reads, without integration in select protooncogenes. These results suggest potential therapeutic benefit of AAV9 PSCGT for neuromuscular disorders but warrant caution for exposure of female germ cells.

Effects of gestational intermittent hypoxia on the autoresuscitation response following anoxia-induced apneas in neonatal mice

Introduction: Sudden infant death syndrome (SIDS) is the leading cause of death in infants from one month to one year of age. Smoking is a significant risk factor in SIDS, and smoking probably causes intermittent hypoxia in the fetus. Therefore, we modeled the effects of intermittent hypoxia during gestation in mice to determine if gestational intermittent hypoxia would adversely affect the capacity of neonatal mice to autoresuscitate effectively after exposure to anoxic stimuli. A degraded capacity to autoresuscitate could explain, in part, why infants born to mothers who smoke have an increased risk of dying from of SIDS. The primary objective of this study was to investigate the effects of intermittent gestational hypoxia on the autoresuscitation responses to anoxia-induced apneas in neonatal mice. Our hypothesis was that pups born to intermittently hypoxic dams would struggle more to recover from anoxic apneas — would have impaired cardiorespiratory responses to anoxic stress compared to the control mice born to dams exposed to room air during gestation. Methods: We studied C57Bl/6 dams who previously had one litter to prove their capability to breed and rear pups. Dams were placed in either an hypoxic or room air chamber at approximately embryonic day 5 (E5; fifth day of pregnancy). The fractional inspired oxygen concentration (FIO2) was reduced to 11% for 10 minutes, three times per hour, with 10 minute periods of 21% oxygen between them. Each hour of intermittent hypoxia was followed by an hour breathing 21% FiO2. The room air control chamber was maintained at a constant FIO2 of 21% with inflows of compressed air that mimicked the noise and flow of nitrogen in the hypoxic chamber. The dams spent the entirety of their pregnancies in the environmental chambers. Once the pups were delivered, the cages were placed in room air, housing environments. The pups were allowed to stay with their mothers until approximately post-natal day 8 (P8). On P8, each pup was fitted into a head out plethysmograph to measure respiratory flow using a pneumotachograph. We recorded respiratory activity, ECG activity, and body movements (using a piezo sensitive film). The pups were maintained at ~34 degrees C in the pneumotachographic chamber. We measured the highest respiratory rate, highest heart rate, longest apnea, gasp latency, mean heart rate recovery, and mean respiratory rate recovery. Results: The mean heart rate recovery and mean longest apnea (in seconds) doubled in the mice exposed to gestational intermittent hypoxia compared to pups born to dams exposed to room air during gestation. The mean heart rate recovery and mean longest apnea in gestationally hypoxic pups was 41.0 +/- 13.7 seconds and 22.05 +/- 12.0 seconds, respectively. The mean heart rate recovery and mean longest apnea in normal, room air-exposed pups was 22.4 +/- 11.1 seconds and 10.3 +/- 7.0 seconds, respectively. The longest apnea in gestational intermittent hypoxic pups was significantly longer than in control pups (53.0 +/- 13.7 seconds and 19.1 +/- 15.0 seconds). Pups exposed to gestational intermittent hypoxia also had increased mean respiratory rate recovery times and gasp latency times (start of apnea until the first gasp). The average body weight of hypoxic pups was 4.3 grams while the average body weight for the normal pups was 4.9 grams. Conclusion: Gestational intermittent hypoxia was associated with delayed and prolonged the autoresuscitation responses in neonatal mice following acute anoxic exposure. We hypothesize that this is due to changes in the brain’s serotonin system. We are investigating how intrauterine hypoxia may decrease serotonin and serotonergic signaling pathways as changes in serotonergic function may degrade the capacity of mice to autoresuscitate following anoxic gas exposure. NIH/NICHD. HD100823 and NIH/NICHD. HD 107060. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of in ovo feeding of xylobiose and xylotriose on growth performance, carcass traits, ileal histomorphometry, and immune-related gene expression in broiler chickens

Xylooligosaccharides (XOS) are known as prebiotics which promote beneficial gut microbes, improve gut health and the performance of broilers. Xylobiose (XOS2) and xylotriose (XOS3) are two compounds in the XOS group. In ovo feeding could deliver these prebiotics to the embryo, and this is considered as an effective method for studying the effects of the prebiotics during embryonic development and the post-hatch period. This study investigated the effects of in ovo feeding of XOS2 and XOS3 on growth performance, carcass traits, ileal histomorphometry, and immune-related gene expression in broilers. A total of 144 fertile eggs of Cobb 500 broilers were divided into three groups: (a) non-injected control (CON), (b) XOS2, and (c) XOS3. The eggs from XOS2 and XOS3 groups were injected with 0.5ml of 0.85% normal saline solution containing 3mg of XOS2 and 3mg of XOS3, respectively, into their amniotic sac on the 17th embryonic day. A total of 131 hatched chicks were raised in 18 replicate pens (5 or more chicks per pen) following standard management practices and fed a corn-soybean meal-based diet for 21 days. The feed intake and body weight (BW) data were recorded weekly. The chickens were euthanized on day 7, day 14, and day 21 to collect ileum sections, and the carcass traits were recorded on only day 21. Total RNA was extracted from ileum tissues, and qPCR was performed to measure the gene expression. All the statistical analyses considered the significance level at a P value <0.05. The result showed that in ovo treatments did not affect the hatchability of the eggs. The XOS2 group showed a trend to increase the BW on day 0, compared to CON (P=0.052), and the XOS3 group improved FCR than the XOS2 group in the first week. There was no difference (P>0.05) among the groups for the weight of the breast muscle, drumsticks, gizzard, proventriculus, and carcass weight at the age of 21 days. The histomorphometry of the ileum on day 7 revealed a significant reduction (P<0.05) of villi height (VH), villi width (VW), and crypt depth (CD) in XOS2, compared to the CON group. On day 7, a lower CD and a higher VH to CD ratio were recorded in XOS3, compared to CON. On day 14, XOS3 showed an increase in VW and villi surface area (VSA) compared to XOS2 birds. On day 7, the expression of IL10 in XOS2 was higher compared to CON and XOS3, and CD56 was significantly higher (P<0.05) in the XOS2 compared to CON group. On day 21, the XOS2 and XOS3 significantly lower the expression of IFNγ. Overall, XOS2 and XOS3 significantly increased immunity until day 21. In conclusion, in ovo XOS demonstrated the potential to modulate gut morphology and immunity of the chickens during the post-hatch days.

Selective Timing of Maternal Immune Activation Alters Developmental Milestones, Behavior, and Blood-Brain Barrier Integrity

Background and Hypothesis: Maternal immune activation (MIA) has been associated with increased risk for neuropsychiatric disease. Fetal exposure to maternal infection activates different toll-like receptors (TLRs) to initiate innate inflammatory responses in mother and fetus. The goal of the current study is to determine sex-dependent aspects of MIA during mid-gestation on neurodevelopment. Methods: Timed-pregnant female mice were administered RQ (TLR7 agonist) or vehicle saline on embryonic day (E) 12.5. Maternal and fetal cytokines were measured to ensure infection of pregnant dam (E15). Offspring were assessed postnatally for developmental milestones and behavior. Adult offspring were perfused with fluorescein isothiocyanate (FITC; 100mg/mL) to visualize blood vessel integrity (FITC leakage; Frahm &amp; Tobet, 2015). To determine neural molecular mechanisms for behavioral changes, sections through the paraventricular nucleus of the hypothalamus (PVN) of offspring were immunolabeled for Glial Fibrillary Acidic Protein (GFAP; astrocytic end feet), Desmin (pericytes) and IBA-1 (microglia). Results: Maternal cytokines IL-6 (p&lt;0.05) and IL-10 (p&lt;0.01) were higher while TNFα (p&lt;0.01) and IL-17 were lower 3 days after RQ-exposure. In fetuses (E15), IL-6 (p&lt;0.05) and IL-17 (p&lt;0.0001) were greater, while IL-10 was higher only in males (p&lt;0.001) exposed to maternal infection. RQ-exposed males also had reduced TNFα (p&lt;0.05). Additionally, RQ-exposed offspring had lower body weights at birth and delayed eye openings. Females exposed to maternal RQ exhibited slower onset of puberty with delayed vaginal openings. On the behavioral side, juvenile and adult offspring of RQ injected moms exhibited less social-like behavior (p&lt;0.001 for all groups) with lower hedonic-like behavior selectively in females (p&lt;0.001). RQ-offspring further showed greater leakage in the PVN indicated by more FITC in the extravascular space (males p&lt;0.001, females p&lt;0.01). GFAP + (astrocytic end feet) coverage of FITC-labeled vessels was higher in the PVN selectively in RQ males (p&lt;0.001). Desmin+ (pericyte) coverage was greater in the PVN of RQ males (p&lt;0.01) and females (p&lt;0.001). Finally, there was greater number of IBA-1+ cells in the adult offspring PVN of both sexes (p&lt;0.01) after maternal injection of RQ. Conclusions: This study provides support for sex-dependent influences of fetal antecedents for altered brain development and behavioral outputs that could be indicative of increased susceptibility for adult disorders through immune mechanisms. Future studies will examine how timing of infection during gestation (mid vs. late) changes neurodevelopmental outputs in offspring. Supported by ORWH-NIMH U54 SCORE-MH118919. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Single-nucleus transcriptional and chromatin accessible profiles reveal critical cell types and molecular architecture underlying chicken sex determination

IntroductionUnderstanding the sex determination mechanisms in birds has great significance for the biological sciences and production in the poultry industry. Sex determination in chickens is a complex process that involves fate decisions of supporting cells such as granulosa or Sertoli cells. However, a systematic understanding of the genetic regulation and cell commitment process underlying sex determination in chickens is still lacking. ObjectivesWe aimed to dissect the molecular characteristics associated with sex determination in the gonads of chicken embryos. MethodsSingle-nucleus RNA-seq (snRNA-seq) and ATAC-seq (snATAC-seq) analysis were conducted on the gonads of female and male chickens at embryonic day 3.5 (E3.5), E4.5, and E5.5. ResultsHere, we provided a time-course transcriptional and chromatin accessible profiling of gonads during chicken sex determination at single-cell resolution. We uncovered differences in cell composition and developmental trajectories between female and male gonads and found that the divergence of transcription and accessibility in gonadal cells first emerged at E5.5. Furthermore, we revealed key cell-type-specific transcription factors (TFs) and regulatory networks that drive lineage commitment. Sex determination signaling pathways, dominated by BMP signaling, are preferentially activated in males during gonadal development. Further pseudotime analysis of the supporting cells indicated that granulosa cells were regulated mainly by the TEAD gene family and that Sertoli cells were driven by the DMRT1 regulons. Cross-species analysis suggested high conservation of both cell types and cell-lineage-specific TFs across the six vertebrates. ConclusionsOverall, our study will contribute to accelerating the development of sex manipulation technology in the poultry industry and the application of chickens as a unique model for studying cell fate decisions.

A Timeline of Cardiac Maturation

Background: Much is known about the differences between adult and immature fetal cardiac myocytes. For example, the switch from glycolysis to oxidative phosphorylation, the increase in sarcomere distance and cell size, along with changes in mitochondrial structure and organization are all known to occur at some point between late embryonic development and adulthood. However, the precise timing of when these changes take place is ill-defined. Here, we report in high temporal resolution when hallmarks of maturation develop to provide a timeline and the order of events that are necessary for structural and physiological cardiomyocyte maturation. Methods and Results: Using murine samples from embryonic day 15 all the way to adulthood, we performed characterization of basic organelles, contractile machinery, and extracellular matrix composition, all shown to be necessary for cardiac maturation. We observed clear differences in when maturation hallmarks became established. Features that increased continuously include growth of the size of the heart and cardiomyocytes. Accompanying cell size changes, an increase in endothelial cell count was observed, although cardiomyocyte growth continued after the endothelial cell population reached a plateau. Seahorse assays revealed that mitochondrial respiration increased continuously over time for Complex 1 and 2 until about postnatal day 15, with complex 4 being relatively stable throughout maturation. Lipid droplets appeared at a higher frequency in ventricular tissue starting at postnatal day 5, coinciding with an increase in sarcomere length. T-tubules began to appear at postnatal day 7 alongside changes in calcium handling genes. Mitochondrial size was found to increase rapidly at postnatal day 19. Discussion: Overall, we found that multiple stages of development seem important to target for enhancing maturation as not all changes occur in a linear fashion. Ultimately, knowledge of when maturational hallmarks develop might inform strategies to enhance maturation of human induced pluripotent stem cell derived cardiomyocytes. T32 Minnesota Muscle Training Grant; Summer’s Wish; Regenerative Medicine Minnesota; Stem Cell Institute Predoctoral Infuse Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Gestational hypothyroxinemia induces ASD-like phenotypes in behavior, proinflammatory markers, and glutamatergic protein expression in mouse offspring of both sexes.

The prevalence of autism spectrum disorder (ASD) has significantly risen in the past three decades, prompting researchers to explore the potential contributions of environmental factors during pregnancy to ASD development. One such factor of interest is gestational hypothyroxinemia (HTX), a frequent condition in pregnancy associated with cognitive impairments in the offspring. While retrospective human studies have linked gestational HTX to autistic traits, the cellular and molecular mechanisms underlying the development of ASD-like phenotypes remain poorly understood. This study used a mouse model of gestational HTX to evaluate ASD-like phenotypes in the offspring. To induce gestational HTX, pregnant mice were treated with 2-mercapto-1-methylimidazole (MMI), a thyroid hormones synthesis inhibitor, in the tap-drinking water from embryonic days (E) 10 to E14. A separate group received MMI along with a daily subcutaneous injection of T4, while the control group received regular tap water during the entire pregnancy. Female and male offspring underwent assessments for repetitive, anxious, and social behaviors from postnatal day (P) 55 to P64. On P65, mice were euthanized for the evaluation of ASD-related inflammatory markers in blood, spleen, and specific brain regions. Additionally, the expression of glutamatergic proteins (NLGN3 and HOMER1) was analyzed in the prefrontal cortex and hippocampus. The HTX-offspring exhibited anxious-like behavior, a subordinate state, and impaired social interactions. Subsequently, both female and male HTX-offspring displayed elevated proinflammatory cytokines in blood, including IL-1β, IL-6, IL-17A, and TNF-α, while only males showed reduced levels of IL-10. The spleen of HTX-offspring of both sexes showed increased Th17/Treg ratio and M1-like macrophages. In the prefrontal cortex and hippocampus of male HTX-offspring, elevated levels of IL-17A and reduced IL-10 were observed, accompanied by increased expression of hippocampal NLGN3 and HOMER1. All these observations were compared to those observed in the Control-offspring. Notably, the supplementation with T4 during the MMI treatment prevents the development of the observed phenotypes. Correlation analysis revealed an association between maternal T4 levels and specific ASD-like outcomes. This study validates human observations, demonstrating for the first time that gestational HTX induces ASD-like phenotypes in the offspring, highlighting the need of monitoring thyroid function during pregnancy.

Mapping Lung Hematopoietic Progenitors: Developmental Kinetics and Response to Influenza A Infection.

The bone marrow is a specialised niche responsible for the maintenance of hematopoietic stem and progenitor cells during homeostasis and inflammation. Recent studies however have extended this essential role to the extramedullary and extravascular lung microenvironment. Here, we provide further evidence for a reservoir of hematopoietic stem and progenitor cells within the lung from embryonic day 18.5 until adulthood. These lung progenitors display distinct microenvironment-specific developmental kinetics compared to their bone marrow counterparts, exemplified by a rapid shift from a common myeloid to megakaryocyte-erythrocyte progenitor dominated niche with increasing age. In adult mice, Influenza A viral infection results in a transient reduction in multipotent progenitors within the lungs, with a parallel increase in downstream granulocyte-macrophage progenitors and dendritic cell populations associated with acute viral infections. Our findings suggest lung hematopoietic progenitors play a role in re-establishing immunological homeostasis in the respiratory mucosa, which may have significant clinical implications for maintaining pulmonary health following inflammatory perturbation.