E14 Embryos Research Articles

Tunneling nanotube-driven complete regeneration of murine fetal skin

This study investigated the three-dimensional (3D) cellular interactions and tunneling nanotubes (TNTs) during fetal mouse skin regeneration on embryonic days 13 (E13) and 15 (E15). We aimed to understand spatial relationships among cell types involved in skin regeneration and assess the potential role of TNTs. Full-thickness skin incisions were performed in E13 and E15 embryos. Wound sites were collected, embedded in epoxy resin, processed for 3D reconstruction (1 μm thickness sections), and subjected to whole-mount immunostaining. We conducted in vitro co-culture experiments with fetal macrophages and fibroblasts to observe TNT formation. To assess the effect of TNTs on skin regeneration, an inhibiting agent (cytochalasin B) was administered to amniotic fluid. Results revealed that E13 epidermal keratinocytes interacted with dermal fibroblasts and macrophages, facilitating skin regrowth. TNT structures were observed at the E13-cell wound sites, among macrophages, and between macrophages and fibroblasts, confirmed through in vitro co-culture experiments. In vitro and utero cytochalasin B administration hindered those formation and inefficient skin texture regeneration at E13 wound sites. This emphasizes the necessity of 3D cellular interactions between epidermal and dermal cells during skin regeneration in mouse embryos at E13. The prevalence of TNT structures indicated their involvement in achieving complete skin texture restoration.

SAT591 Abnormal Levels Of Thyroid Hormones During Development Alters The Migration And Activity Of GnRH Reproductive Neurons

Abstract Disclosure: C. Quignon: None. A. Backer: None. S. Wray: None. Clinical studies have shown that thyroid hormones disorders and alteration of thyroid hormone production by environmental endocrine disruptors, have deleterious effects on reproduction. By acting in the brain on the hypothalamic component of the reproductive axis, abnormal levels of thyroid hormones can disturb sex hormones production, oestrus cycles or puberty onset, possibly leading to subfertility. Moreover, in animal models, endocrine disruptors and thyroid hormones have been shown to have long-lasting transgenerational effects with the maternal hormones influencing the development of foetal reproductive functions. Despite clear evidence of an interaction between the thyroid and reproductive systems, the effect of T3, the active form of thyroid hormones, on the GnRH neurons controlling reproduction, have been poorly studied. In this work we investigated how T3 functionally interacts with GnRH neurons and how abnormal concentration of T3 alters the migration of these neurons during development. Calcium imaging was used to study the direct effect of T3 on GnRH neuronal activity in an ex vivo nasal explants model. Acute administration of T3 (30nM) was found to stimulate the activity of GnRH cells. Dual labelling of GnRH and thyroid hormone receptors (TR) showed that the GnRH neurons express both nuclear and membrane receptors. However, the use of an antagonist that specifically blocks the TR nuclear receptors (1-850), didn’t inhibit the T3 stimulatory effect. In contrast, blockage of integrin αV/β3 membrane receptors (with cilengitide), which have been shown to be activated by T3, prevented the T3-induced increase in GnRH neuronal activity. During development GnRH neurons migrate from the nasal placodes into the brain. Acute administration of T3 for 1hr significatively decreased the migration rate of the GnRH neurons in our ex vivo model. To assess the effect of thyroid disruption during pregnancy on the development of reproductive axis in utero, pregnant females mice have been treated with methimazole to induce hypothyroidism from gestational day 6 to 13. E13 embryos have been collected from treated and control dams and the distribution of migrating GnRH neurons will be compared between both groups. Together these studies are the first to report a direct effect of thyroid hormones on GnRH neuronal activity, through the integrin αV/β3 membrane receptors and show that T3 can modulate the migration of GnRH neurons during development. These results will bring new insights on how thyroid disruption by endocrine disruptor chemicals or thyroid diseases, especially during foetal development, can lead to long term reproductive defects. Presentation: Saturday, June 17, 2023

Hypertrophy of Adipose Tissues in Quail Embryos by in ovo Injection of All-Trans Retinoic Acid.

Excessive adipose accretion causes health issues in humans and decreases feed efficiency in poultry. Although vitamin A has been known to be involved in adipogenesis, effects of all-trans retinoic acid (atRA), as a metabolite of vitamin A, on embryonic adipose development have not been studied yet. Avian embryos are developing in confined egg environments, which can be directly modified to study effects of nutrients on embryonic adipogenesis. With the use of quail embryos, different concentrations of atRA (0 M to 10 μM) were injected in ovo at embryonic day (E) 9, and adipose tissues were sampled at E14. Percentages of fat pad weights in embryo weights were significantly increased in the group injected with 300 nM of atRA. Also, among three injection time points, E5, E7, or E9, E7 showed the most significant increase in weight and percentage of inguinal fat at E14. Injection of atRA at E7 increased fat cell size in E14 embryos with up-regulation of pro-adipogenic marker genes (Pparγ and Fabp4) and down-regulation of a preadipocyte marker gene (Dlk1) in adipose tissues. These data demonstrate that atRA promotes hypertrophic fat accretion in quail embryos, implying important roles of atRA in embryonic development of adipose tissues.

Expression Signatures of microRNAs and Their Targeted Pathways in the Adipose Tissue of Chickens during the Transition from Embryonic to Post-Hatch Development.

As the chick transitions from embryonic to post-hatching life, its metabolism must quickly undergo a dramatic switch in its major energy source. The chick embryo derives most of its energy from the yolk, a lipid-rich/carbohydrate-poor source. Upon hatching, the chick’s metabolism must then be able to utilize a lipid-poor/carbohydrate-rich source (feed) as its main form of energy. We recently found that a number of hepatically-expressed microRNAs (miRNAs) help facilitate this shift in metabolic processes in the chick liver, the main site of lipogenesis. While adipose tissue was initially thought to mainly serve as a lipid storage site, it is now known to carry many metabolic, endocrine, and immunological functions. Therefore, it would be expected that adipose tissue is also an important factor in the metabolic switch. To that end, we used next generation sequencing (NGS) and real-time quantitative PCR (RT-qPCR) to generate miRNome and transcriptome signatures of the adipose tissue during the transition from late embryonic to early post-hatch development. As adipose tissue is well known to produce inflammatory and other immune factors, we used SPF white leghorns to generate the initial miRNome and transcriptome signatures to minimize complications from external factors (e.g., pathogenic infections) and ensure the identification of bona fide switch-associated miRNAs and transcripts. We then examined their expression signatures in the adipose tissue of broilers (Ross 708). Using E18 embryos as representative of pre-switching metabolism and D3 chicks as a representative of post-switching metabolism, we identified a group of miRNAs which work concordantly to regulate a diverse but interconnected group of developmental, immune and metabolic processes in the adipose tissue during the metabolic switch. Network mapping suggests that during the first days post-hatch, despite the consumption of feed, the chick is still heavily reliant upon adipose tissue lipid stores for energy production, and is not yet efficiently using their new energy source for de novo lipid storage. A number of core master regulatory pathways including, circadian rhythm transcriptional regulation and growth hormone (GH) signaling, likely work in concert with miRNAs to maintain an essential balance between adipogenic, lipolytic, developmental, and immunological processes in the adipose tissue during the metabolic switch.

Ontogeny of renin gene expression in the chicken, Gallus gallus

Renin or a renin-like enzyme evolved in ancestral vertebrates and is conserved along the vertebrate phylogeny. The ontogenic development of renin, however, is not well understood in nonmammalian vertebrates. We aimed to determine the expression patterns and relative abundance of renin mRNA in pre- and postnatal chickens (Gallus gallus, White Leghorn breed). Embryonic day 13 (E13) embryos show renal tubules, undifferentiated mesenchymal structures, and a small number of developing glomeruli. Maturing glomeruli are seen in post-hatch day 4 (D4) and day 30 (D30) kidneys, indicating that nephrogenic activity still exists in kidneys of 4-week-old chickens. In E13 embryos, renin mRNA measured by quantitative polymerase chain reaction in the adrenal glands is equivalent to the expression in the kidneys, whereas in post-hatch D4 and D30 maturing chicks, renal renin expressions increased 2-fold and 11-fold, respectively. In contrast, relative renin expression in the adrenals became lower than in the kidneys. Furthermore, renin expression is clearly visible by in situ hybridization in the juxtaglomerular (JG) area in D4 and D30 chicks, but not in E13 embryos. The results suggest that in chickens, renin evolved in both renal and extrarenal organs at an early stage of ontogeny and, with maturation, became localized to the JG area. Clear JG structures are not morphologically detectable in E13 embryos, but are visible in 30-day-old chicks, supporting this concept.

Liver Growth Factor Induces Glia-Associated Neuroprotection in an In Vitro Model of Parkinson´s Disease.

Parkinson’s disease is a neurodegenerative disorder characterized by the progressive death of dopaminergic (DA) neurons in the substantia nigra (SN), which leads to a loss of the neurotransmitter dopamine in the basal ganglia. Current treatments relieve the symptoms of the disease, but none stop or delay neuronal degeneration. Liver growth factor (LGF) is an albumin–bilirubin complex that stimulates axonal growth in the striatum and protects DA neurons in the SN of 6-hydroxydopamine-lesioned rats. Our previous results suggested that these effects observed in vivo are mediated by microglia and/or astrocytes. To determine if these cells are LGF targets, E14 (embryos from Sprague Dawley rats of 14 days) rat mesencephalic glial cultures were used. Treatment with 100 pg/mL of LGF up-regulated the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases 1/2 (ERK1/2) and the cyclic AMP response element binding protein (CREB) phosphorylation in glial cultures, and it increased the microglia marker Iba1 and tumor necrosis factor alpha (TNF-alpha) protein levels. The treatment of E14 midbrain neurons with a glial-conditioned medium from LGF-treated glial cultures (GCM-LGF) prevented the loss of DA neurons caused by 6-hydroxy-dopamine. This neuroprotective effect was not observed when GCM-LGF was applied in the presence of a blocking antibody of TNF-alpha activity. Altogether, our findings strongly suggest the involvement of microglia and TNF-alpha in the neuroprotective action of LGF on DA neurons observed in vitro.

Loss of Dll4 expression in thymic epithelial cell monolayer culture is mediated by reduction in both burst size and burst frequency of its gene transcription

Abstract Commitment of thymic seeding progenitor (TSP) cells to the T-lineage depends on Notch signaling, which requires engagement of the Notch1 receptor on TSPs by the Notch ligand Dll4 on thymic epithelial cells (TECs). We previously showed that when TECs are cultured as monolayers (2-D), Dll4 expression is rapidly lost, as is their ability to support T-cell development in vitro. In the present study, we aim to examine the regulation of Dll4 expression in TECs at the levels of gene transcription and mRNA degradation in TECs from E15 embryos using a combination of multiple techniques. The half-life of Dll4 mRNA in TECs was found, by utilizing quantitative PCR (qPCR) combined with Uridine RNA analog pulse-labeling, only approximately 30 min, which makes its level very sensitive to changes in the rates of gene transcription. Nonetheless, reduction in gene transcription, rather than in mRNA stability, was determined to be the primary molecular mechanism underlying the loss of Dll4 expression in 2-D TECs. FoxN1, the transcription factor that drives thymic epithelial cell development, was found to bind to multiple sites at the Dll4 gene promoter and an enhancer region within its third intron by luciferase reporter assay. Consistent with the luciferase reporter assay result, enforced expression of FoxN1 in 2-D TECs restored Dll4 expression significantly. Further detailed analysis with PrimeFlow RNA assay revealed that FoxN1 regulates only the burst size, but not the burst frequency, of Dll4 gene transcription. We are currently working on identifying the transcription regulator(s) that could bind to the enhancer region to regulate the burst frequency of Dll4 gene transcription.

An agarose gel-based neurosphere culture system leads to enrichment of neuronal lineage cells in vitro

Stem cell-based therapy holds great potential especially for neurological disorders. However, clinical applications await further understanding of many aspects of stem cell differentiation and development of technology enabling manipulation of stem cells into desired cell types in the central nervous system. Here, we developed a new method that leads to enrichment of neuronal lineage cells in neural stem cell cultures. The protocol involves cultivation of primary cells derived from the forebrains of rat E18 embryos above a layer of nonadhesive hard agarose gel in the form of neurospheres. In contrast to the neurospheres that were cultured above an anti-adhesive hydrogel layer, the primary cells that were cultured above a layer of agarose gel preferentially differentiated into β-III tubulin-positive neurons when allowed to undergo differentiation in vitro.In an effort to investigate the mechanism behind this observation, we found that the gene expression of a vertebrate neuronal determination gene (neurogenin1) was enhanced in the neurospheres that proliferated above a layer of agarose gel as compared with the control, and the gene expression level of neurogenin1 was quite well correlated with the rigidity of agarose gel. These results indicate that agarose gel can contribute, at least in part, to enrich neuronal progenitors and immature postmitotic neurons during neurosphere formation and may provide additional information to establish efficient protocols for the neural stem cell-based study.

Histamine Modulates Midbrain Dopamine Neuron Differentiation Through the Regulation of Epigenetic Marks.

During midbrain development, dopamine neuron differentiation occurs before birth. Epigenetic processes such as DNA methylation and demethylation as well as post-translational modification of histones occur during neurogenesis. Here, we administered histamine (HA) into the brain of E12 embryos in vivo and observed significant lower immunoreactivity of Lmx1a+ and Tyrosine Hydroxylase (TH)+ cells, with parallel decreases in the expression of early (Lmx1a, Msx1) and late (Th) midbrain dopaminergic (mDA) genes. With MeDIP assays we found that HA decreases the percentage of 5-methylcytosine of Pitx3 and Th, without changes in 5-hydroxymethylcytosine. Additionally, HA treatment caused a significant increase in the repressive epigenetic modifications H3K9me3 in Pitx3 and Th, and also more H3K27me3 marks in Th. Furthermore, HA has a long-term effect on the formation of the nigrostriatal and mesolimbic/mesocortical pathways, since it causes a significant decrease in midbrain TH immunoreactivity, as well as alterations in dopaminergic neuronal fibers, and significant lower TH-positive area in the forebrain in whole-mount stainings. These findings suggest that HA diminishes dopaminergic gene transcription by altering several epigenetic components related to DNA and histone modifications, which affects mDA neuron progression during development.

P127 THE TRANS-GOLGI NETWORK PROTEIN AFTIPHILIN REGULATES COLONIC EPITHELIAL CELL MIGRATION AND IS CRUCIAL TO EMBRYONIC DEVELOPMENT

Colonic epithelium forms a selective barrier between lumen and colonic mucosa. The integrity of colonic epithelium is maintained by cell proliferation, migration and expression of cellular junctions. A compromised colonic epithelium contributes to colonic inflammation in IBD patients. Previously, we showed that aftiphilin (AFTPH) is significantly downregulated in colonic biopsies from UC patients and involved in colonic epithelial cell permeability. In this study, we examined the role of AFTPH in cell proliferation and migration in vitro and in colonic inflammation by generation of total AFTPH knock-out mice. Human colonic epithelial NCM460 cells was transfected with si-RNA against AFTPH (si-AFTPH) and its control using lipofectamineRNAimax (Thermo Fisher Scientific). Two days post-transfection, cell migration and cell proliferation were assessed by scratch assay and MTT assay, respectively. Hemizygous AFTPH knock-out (AFTPH+/-) mice were generated by crossing heterozygous AFTPH-floxed mice (AFTPHflox/+, Australian Regenerative Medicine Institute, Monash University) and CMV-Cre mice (JAX no. 006054). Total AFTPH knock-out (AFTPH-/-) mice were generated by crossing AFTPH+/- mice. Neonates and embryos from Embryonic day (E) 13 and 17 were genotyped (Transnetyx). Gene-silencing of AFTPH in human colonic epithelial NCM460 cells reduced the rate of cell migration when compared to that of cells transfected with control si-RNA (area ratio of wound closure: 0.1 ± 0.04 vs. 0.2 ± 0.03). On contrary, AFTPH gene silencing did not affect cell proliferation when compared to control cells (OD570nm: 0.5 ± 0.01 vs. 0.5 ± 0.11). To study the role of AFTPH in colonic inflammation and intestinal permeability in vivo, we aimed to generate total AFTPH knock-out (AFTPH-/-) mice by crossbreeding AFTPH+/- mice. However, no AFTPH-/- mice were identified in neonates (AFTPH+/+: AFTPH+/-: AFTPH-/- = 5:16:0, average litter size = 3.0 ± 1.38). To study whether total AFTPH knock-out is embryonic lethal, embryos from E13 and E17 were collected and genotyped. Interestingly, our results showed that 7 AFTPH-/-mice were identified among the E13 embryos (AFTPH+/+: AFTPH+/-: AFTPH-/- = 5:14:7, average litter size of 8.7 ± 1.15); while 1 AFTPH-/- mouse were identified among the E17 embryos (AFTPH+/+: AFTPH+/-: AFTPH-/- = 10:11:1, average litter size of 7.3 ± 0.58). Our results indicated that AFTPH regulates colonic epithelial cell migration, but not cell proliferation, suggesting a potential role in wound healing during colitis. Importantly, total AFTPH knock-out is possibly embryonic lethal. An intestinal epithelial cell-specific (Villin-Cre) AFTPH knock-out mice is now being generated for studying the role of AFTPH in colonic cell function in vivo.

P127 THE TRANS-GOLGI NETWORK PROTEIN AFTIPHILIN REGULATES COLONIC EPITHELIAL CELL MIGRATION AND IS CRUCIAL TO EMBRYONIC DEVELOPMENT

Colonic epithelium forms a selective barrier between lumen and colonic mucosa. The integrity of colonic epithelium is maintained by cell proliferation, migration and expression of cellular junctions. A compromised colonic epithelium contributes to colonic inflammation in IBD patients. Previously, we showed that aftiphilin (AFTPH) is significantly downregulated in colonic biopsies from UC patients and involved in colonic epithelial cell permeability. In this study, we examined the role of AFTPH in cell proliferation and migration in vitro and in colonic inflammation by generation of total AFTPH knock-out mice. Human colonic epithelial NCM460 cells was transfected with si-RNA against AFTPH (si-AFTPH) and its control using lipofectamineRNAimax (Thermo Fisher Scientific). Two days post-transfection, cell migration and cell proliferation were assessed by scratch assay and MTT assay, respectively. Hemizygous AFTPH knock-out (AFTPH+/-) mice were generated by crossing heterozygous AFTPH-floxed mice (AFTPHflox/+, Australian Regenerative Medicine Institute, Monash University) and CMV-Cre mice (JAX no. 006054). Total AFTPH knock-out (AFTPH-/-) mice were generated by crossing AFTPH+/- mice. Neonates and embryos from Embryonic day (E) 13 and 17 were genotyped (Transnetyx). Gene-silencing of AFTPH in human colonic epithelial NCM460 cells reduced the rate of cell migration when compared to that of cells transfected with control si-RNA (area ratio of wound closure: 0.1 ± 0.04 vs. 0.2 ± 0.03). On contrary, AFTPH gene silencing did not affect cell proliferation when compared to control cells (OD570nm: 0.5 ± 0.01 vs. 0.5 ± 0.11). To study the role of AFTPH in colonic inflammation and intestinal permeability in vivo, we aimed to generate total AFTPH knock-out (AFTPH-/-) mice by crossbreeding AFTPH+/- mice. However, no AFTPH-/- mice were identified in neonates (AFTPH+/+: AFTPH+/-: AFTPH-/- = 5:16:0, average litter size = 3.0 ± 1.38). To study whether total AFTPH knock-out is embryonic lethal, embryos from E13 and E17 were collected and genotyped. Interestingly, our results showed that 7 AFTPH-/-mice were identified among the E13 embryos (AFTPH+/+: AFTPH+/-: AFTPH-/- = 5:14:7, average litter size of 8.7 ± 1.15); while 1 AFTPH-/- mouse were identified among the E17 embryos (AFTPH+/+: AFTPH+/-: AFTPH-/- = 10:11:1, average litter size of 7.3 ± 0.58). Our results indicated that AFTPH regulates colonic epithelial cell migration, but not cell proliferation, suggesting a potential role in wound healing during colitis. Importantly, total AFTPH knock-out is possibly embryonic lethal. An intestinal epithelial cell-specific (Villin-Cre) AFTPH knock-out mice is now being generated for studying the role of AFTPH in colonic cell function in vivo.

Prenatal Sensory Stimulation Induces BDNF Gene Expression in the Brain and Potentiates the Development of Species-Specific Predisposition in Newborn Chicks.

We studied the effects of light and non-specific sound stimulation of domestic chick embryos on their filial preference as well as on the expression of two transcriptional factors c-Fos and Egr-1 and neurotrophin BDNF in the embryo brain. Prenatal light stimulation increased preference of the "natural" object, thus producing a priming effect. In the brain of E19 embryos, c-Fos and Egr-1 were expressed at a high basal level and neither light nor sound stimulation affected the number of cells expressing these factors. BDNF mRNA was also present in a number of brain areas of non-stimulated embryos, but light and sound stimulation enhanced the expression of BDNF mRNA in brain structures associated with filial imprinting. These findings suggest that BDNF is probably involved in the effects of prenatal priming on the development of species-specific behavior.

Geldanamycin administration reduces the amount of primordial germ cells in the mouse embryo

Heat shock proteins (HSPs) are expressed or overexpressed in response to exposure to stress. They act as molecular chaperones, ensuring the correct folding of numerous client proteins. HSP90 is one of the most conserved HSPs. Its role extends beyond stress tolerance. HSP90 also contributes to development, differenciation, apoptosis and oncogenesis. Numerous tumors are associated with an overexpression of HSP90 and this expression can be used to evaluate its metastatic capacity. Primordial germ cells (PGCs) exhibit HSP90 expression under normal conditions. PGCs arise early in development and migrate by a combination of passive and active movements towards the gonads. The aim of this work was to study the impact of an inhibition of HSP90 on the migration of the PGCs. Geldanamycin, a well established HSP90 inhibitor with potent antitumor properties was used to achieve this inhibition. 5mg of Geldanamycin were administered to E8 pregnant mice. E17 embryos were removed and fixed for staining and Immunohistochemistry with anti-HSP90 and anti-VASA antibodies. Geldanamycin-treated mouse embryos exhibited less VASA-immunopositive cells compared to the non-treated ones. These results suggest that geldanamycin administration at the time of PGCs migration reduces the number of PGCs in the gonads. HSP90 and VASA stainings were identical. We therefore expressed the idea that HSP90 could be used as a reliable marker for PGCs.

Role of Central Hypercapnia in Rhythmic Spontaneous Neural Activity and Breathing Rhythms in the Developing Avian Medulla

The execution of rhythmic behaviors such as breathing requires proper construction of neural circuits during embryogenesis and throughout the perinatal period. A unique feature of developing circuits that may support neuronal circuit organization is the appearance and maintenance of rhythmic spontaneous neural activity (rSNA). Although rSNA has been studied extensively using a variety of in vitro preparations, rarely has rSNA been observed continuously from its onset until it contributes to mature behaviors. Little is known about how rSNA is maintained throughout the embryonic and perinatal periods due to difficulties accessing mammalian embryos and the delicate nature of embryonic tissue. To better understand these details, we used the zebra finch embryo brainstem preparation, and recorded rSNA and subsequent breathing rhythms continuously from embryonic day (E) 4 through hatching on E14. Our objective was to test whether breathing‐related rhythms during the aqueous embryonic period and aerial breathing rhythms during the perinatal period are sensitive to changes in CO2 (hypercapnia or hypocapnia). We hypothesized that hypercapnic and hypocapnic stimuli would alter rSNA frequency according to embryonic age. We predicted prenatal embryos (<E11) would show changes in rSNA that corresponded with generalized dampening or strengthening of neural activity based on whether pH was more acidic or alkaline, respectively. In non‐respiratory neurons, acidic stimuli typically lower activity and alkaline stimuli raise it. In contrast, we predicted the most mature, and continuously air breathing, embryos (>E11) would show respiratory‐related directional alterations in breathing‐related rhythm frequency similar to neonatal mammals exposed to hypercapnia or hypocapnia. To test our hypotheses, we superfused zebra finch brainstems with artificial cerebrospinal fluid equilibrated with control (90% O2, 5% CO2), hypercapnic (90% O2, 8% CO2), or hypocapnic (90% O2, 2% CO2) gases. During the hour‐long treatments, rSNA (<E11) or breathing‐related rhythms (>E11) were recorded from the spinal accessory nerve. Results suggest that CO2 modifies rSNA in younger embryos as well as breathing rhythms in older embryos, however the polarity of the response reversed near E11. Specifically, hypercapnia decreased rSNA frequency up to approximately 66 ± 14% of control frequency in prenatal embryos (<E11) and increased respiratory‐related neural activity frequency to approximately 112 ± 10% of control in perinatal embryos (>E11). Moreover, preliminary data from experiments where [NaHCO3] was raised such that pH was 7.7 show that hypocapnia increased the frequency of rSNA in E4–E11 embryos and leads to slow, irregular breathing‐related rhythms in E11–E14 embryos. These data indicate that the undifferentiated future respiratory circuits of the developing embryo are capable of sensing changes in CO2 and that this signal may serve to regulate rhythmic oscillations prior to air breathing. As predicted, near E11, when air breathing begins, the response to CO2 switches from the embryonic condition to the mature condition—consistent with CO2 effects in perinatal and newborn rodents and humans.Support or Funding InformationIdaho IDeA Network of Biomedical Research and Excellence (INBRE) Program Grant # P20 GM103408 (National Institute of General Medical Sciences) to JRW and CWL, and the National Institute of Neurological Disorders and Stroke via the Health Academic Research Enhancement Award (AREA) Program Grant # 1R15NS087521‐01A1 to JQP.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glucocorticoid Receptor Signaling Is Not Required for In Vivo Adipogenesis.

Regulation of adipogenesis is of major interest given that adipose tissue expansion and dysfunction are central to metabolic syndrome. Glucocorticoids (GCs) are important for adipogenesis in vitro. However, establishing a role for GCs in adipogenesis in vivo has been difficult. GC receptor (GR)‒null mice die at birth, a time at which wild-type (WT) mice do not have fully developed white adipose depots. We conducted several studies aimed at defining the role of GC signaling in adipogenesis in vitro and in vivo. First, we showed that GR-null mouse embryonic fibroblasts (MEFs) have compromised ability to form adipocytes in vitro, a phenotype that can be partially rescued with a peroxisome proliferator-activated receptor γ agonist. Next, we demonstrated that MEFs are capable of forming de novo fat pads in mice despite the absence of GR or circulating GCs [by bilateral adrenalectomy (ADX)]. However, ADX and GR-null fat pads and their associated adipocyte areas were smaller than those in controls. Second, using adipocyte-specific luciferase reporter mice, we identified adipocytes in both WT and GR-null embryonic day (E)18 mouse embryos. Lastly, positive perilipin staining in WT and GR-null E18 embryos confirmed the presence of early white inguinal and brown adipocytes. Taken together, these results provide compelling evidence that GCs and GR augment but are not required for the development of functional adipose tissue in vivo.

Characterization of Superficial Dorsal Horn Neurons from “Tamamaki” Mice and Stability of their GAD67-EGFP Phenotype in Defined-Medium Organotypic Culture

Defined medium organotypic cultures (DMOTC) containing spinal dorsal horn neurons are especially useful in studying the etiology and pharmacology of chronic pain. We made whole-cell recordings from neurons in acutely isolated mouse spinal cord slices or from those maintained in DMOTC for up to 6 weeks. In acute slices, neurons in the substantia gelatinosa exhibited 7 different firing patterns in response to 800-ms depolarizing current commands; delay (irregular), delay (tonic), tonic, regular firing, phasic, initial bursting and single spiking. Initial bursting and regular firing neurons are not found in rat substantia gelatinosa. In acute slices from “Tamamaki” mice that express enhanced green fluorescent protein (EGFP) under the control of the glutamic acid decarboxylase 67 (GAD67) promotor, tonic, phasic and regular firing neurons exhibited the strongest GABAergic (GAD67-EGFP+) phenotype. Delay (tonic) and delay (irregular) neurons almost never expressed GAD67 (GAD67-EGFP−) and are likely glutamatergic. All seven phenotypes were preserved in mouse spinal cord neurons in DMOTC prepared from e12 embryos and the GAD67-EGFP+ phenotype continued to associate with phasic and regular firing neurons. Only 3 out of 51 GAD67-EGFP+ neurons exhibited a delay (tonic) firing pattern. Modifications to the mouse genome thus continue to be expressed when embryonic neurons develop in vitro in DMOTC. However, analysis of the amplitude and interevent interval of spontaneous EPSCs (sEPSCs) indicated substantial re-arrangement of synaptic connections within the cultures. Despite this, the characteristics and age-dependence of asynchronous oscillatory activity, as monitored by multiphoton Ca2+ imaging, were similar in acute slices and in DMOTC.

Newly raised anti-VAChT and anti-ChAT antibodies detect cholinergic cells in chicken embryos.

The autonomic nervous system consists of sympathetic and parasympathetic nerves, which functionally antagonize each other to control physiology and homeostasis of organs. However, it is largely unexplored how the autonomic nervous system is established during development. In particular, early formation of parasympathetic network remains elusive because of its complex anatomical structure. To distinguish between parasympathetic (cholinergic) and sympathetic (adrenergic) ganglia, vesicular acetylcholine transporter (VAChT) and choline O-acetyltransferase (ChAT), proteins associated with acetylcholine synthesis, are known to be useful markers. Whereas commercially available antibodies against these proteins are widely used for mammalian specimens including mice and rats, these antibodies do not work satisfactorily in chickens, although chicken is an excellent model for the study of autonomic nervous system. Here, we newly raised antibodies against chicken VAChT and ChAT proteins. One monoclonal and three polyclonal antibodies for VAChT, and one polyclonal antibody for ChAT were obtained, which were available for Western blotting analyses and immunohistochemistry. Using these verified antibodies, we detected cholinergic cells in Remak ganglia of autonomic nervous system, which form in the dorsal aspect of the digestive tract of chicken E13 embryos. The antibodies obtained in this study are useful for visualization of cholinergic neurons including parasympathetic ganglia.

AT2 R deficiency mediated podocyte loss via activation of ectopic hedgehog interacting protein (Hhip) gene expression.

Angiotensin II type 2 receptor (AT2 R) deficiency in AT2 R knockout (KO) mice has been linked to congenital abnormalities of the kidney and urinary tract; however, the mechanisms by which this occurs are poorly understood. In this study, we examined whether AT2 R deficiency impaired glomerulogenesis and mediated podocyte loss/dysfunction in vivo and in vitro. Nephrin-cyan fluorescent protein (CFP)-transgenic (Tg) and Nephrin/AT2 RKO mice were used to assess glomerulogenesis, while wild-type and AT2 RKO mice were used to evaluate maturation of podocyte morphology/function. Immortalized mouse podocytes (mPODs) were employed for in vitro studies. AT2 R deficiency resulted in diminished glomerulogenesis in E15 embryos, but had no impact on actual nephron number in neonates. Pups lacking AT2 R displayed features of renal dysplasia with lower glomerular tuft volume and podocyte numbers. In vivo and in vitro studies demonstrated that loss of AT2 R was associated with elevated NADPH oxidase 4 levels, which in turn stimulated ectopic hedgehog interacting protein (Hhip) gene expression in podocytes. Consequently, ectopic Hhip expression activation either triggers caspase-3 and p53-related apoptotic processes resulting in podocyte loss, or activates TGFβ1-Smad2/3 cascades and α-SMA expression to transform differentiated podocytes to undifferentiated podocyte-derived fibrotic cells. We analyzed HHIP expression in the kidney disease database (Nephroseq) and then validated this using HHIP immunohistochemistry staining of human kidney biopsies (controls versus focal segmental glomerulosclerosis). In conclusion, loss of AT2 R is associated with podocyte loss/dysfunction and is mediated, at least in part, via augmented ectopic Hhip expression in podocytes. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Involvement of posttranscriptional regulation of Clock in the emergence of circadian clock oscillation during mouse development

Circadian clock oscillation emerges in mouse embryo in the later developmental stages. Although circadian clock development is closely correlated with cellular differentiation, the mechanisms of its emergence during mammalian development are not well understood. Here, we demonstrate an essential role of the posttranscriptional regulation of Clock subsequent to the cellular differentiation for the emergence of circadian clock oscillation in mouse fetal hearts and mouse embryonic stem cells (ESCs). In mouse fetal hearts, no apparent oscillation of cell-autonomous molecular clock was detectable around E10, whereas oscillation was clearly visible in E18 hearts. Temporal RNA-sequencing analysis using mouse fetal hearts reveals many fewer rhythmic genes in E10-12 hearts (63, no core circadian genes) than in E17-19 hearts (483 genes), suggesting the lack of functional circadian transcriptional/translational feedback loops (TTFLs) of core circadian genes in E10 mouse fetal hearts. In both ESCs and E10 embryos, CLOCK protein was absent despite the expression of Clock mRNA, which we showed was due to Dicer/Dgcr8-dependent translational suppression of CLOCK. The CLOCK protein is required for the discernible molecular oscillation in differentiated cells, and the posttranscriptional regulation of Clock plays a role in setting the timing for the emergence of the circadian clock oscillation during mammalian development.

Interleukin-10 inhibits neuroinflammation-mediated apoptosis of ventral mesencephalic neurons via JAK-STAT3 pathway

Neuroinflammation plays an important role in the pathogenesis of Parkinson's disease. Interleukin (IL)-10 is one of the most important and best anti-inflammatory cytokines. The objective of this report is to investigate whether IL-10 has any role in protecting ventral mesencephalic (VM) neurons in in vitro model of neuroinflammation. In this study, primary neuron-enriched culture was prepared from the VM tissues of E14 embryos of rats. The cells were pretreated with IL-10 (15 or 50ng/mL) for 1h followed by lipopolysaccharide (LPS, 50ng/mL) application. We found LPS induced neuronal apoptosis and loss while pretreatment with IL-10 reduced neuronal damage after exposure of LPS toxicity. Furthermore, signal transduction pathways related to IL-10 in VM neurons were studied in inflammatory condition. We used both shRNA and pharmacologic inhibition to determine the role of the IL-10 receptor (IL-10R) and its downstream signaling pathways in LPS-induced VM neuronal toxicity. Silence of the IL-10R gene in VM neurons abolished IL-10 mediated protection and the properties of anti-inflammatory and anti-apoptosis. IL-10 also induced phosphorylation of signal transducer and activator of transcription (STAT) 3 in VM neurons. Pretreatment with the specific Janus kinase (JAK) inhibitor reduced STAT3 phosphorylation and blocked IL-10 mediated protection against LPS. These findings suggest that IL-10 provides neuroprotection by acting via IL-10R and its down-stream JAK-STAT3 signal pathways in VM neurons.