Mouse embryo CoCoPUTs: novel murine transcriptomic-weighted usage website featuring multiple strains, tissues, and stages.

Mouse (Mus musculus) models have been heavily utilized in developmental biology research to understand mammalian embryonic development, as mice share many genetic, physiological, and developmental characteristics with humans. New explorations into the integration of temporal (stage-specific) and transcriptional (tissue-specific) data have expanded our knowledge of mouse embryo tissue-specific gene functions. To better understand the substantial impact of synonymous mutational variations in the cell-state-specific transcriptome on a tissue's codon and codon pair usage landscape, we have established a novel resource-Mouse Embryo Codon and Codon Pair Usage Tables (Mouse Embryo CoCoPUTs). This webpage not only offers codon and codon pair usage, but also GC, dinucleotide, and junction dinucleotide usage, encompassing four strains, 15 murine embryonic tissue groups, 18 Theiler stages, and 26 embryonic days. Here, we leverage Mouse Embryo CoCoPUTs and employ the use of heatmaps to depict usage changes over time and a comparison to human usage for each strain and embryonic time point, highlighting unique differences and similarities. The usage similarities found between mouse and human central nervous system data highlight the translation for projects leveraging mouse models. Data for this analysis can be directly retrieved from Mouse Embryo CoCoPUTs. This cutting-edge resource plays a crucial role in deciphering the complex interplay between usage patterns and embryonic development, offering valuable insights into variation across diverse tissues, strains, and stages. Its applications extend across multiple domains, with notable advantages for biotherapeutic development, where optimizing codon usage can enhance protein expression; one can compare strains, tissues, and mouse embryonic stages in one query. Additionally, Mouse Embryo CoCoPUTs holds great potential in the field of tissue-specific genetic engineering, providing insights for tailoring gene expression to specific tissues for targeted interventions. Furthermore, this resource may enhance our understanding of the nuanced connections between usage biases and tissue-specific gene function, contributing to the development of more accurate predictive models for genetic disorders.

Morphological features of the mouse duodenocolic fold in foetus and adult.

For the mechanism of duodenojejunal flexure (DJF) morphogenesis in mice, we consider the gut tube itself and the gut mesentery as important players. In this study, we focussed on the morphological features of the gut mesentery around the mouse duodenum, especially the duodenocolic fold at embryonic day (E) 18.5 and the adult phase. The duodenocolic fold, a sheet of the mesentery, was located between the entire ascending duodenum and the descending colon. At E18.5, in the cranial area near the DJF, the duodenocolic fold joined both the mesocolon and the mesojejunal part of the root of the mesentery. In the middle and caudal areas, the duodenocolic fold joined the mesocolon. Interestingly, along with the ascending duodenum, the duodenocolic fold contained a smooth muscle bundle. The smooth muscle bundle continued from the outer muscular layer of the middle to the caudal part of the ascending duodenum. The three-dimensional imaging of the foetal duodenocolic fold revealed that the smooth muscle bundle had short and long apexes towards the proximal and distal parts of the root of the mesentery, respectively. At the adult phase, the duodenocolic fold had a much thinner connective tissue with a larger surface area in comparison with the duodenocolic fold at E18.5. The adult duodenocolic fold also contained the smooth muscle bundle which was similar to the foetal duodenocolic fold. A part of the duodenocolic fold connecting to the mesojejunal part of the root of the mesentery seemed to be homologous to the superior duodenal fold in humans, known as the duodenojejunal fold; by contrast, most of the duodenocolic fold seemed to be homologous to the inferior duodenal fold in humans, known as the duodenomesocolic fold. The smooth muscle bundle in the mouse duodenocolic fold seemed to play a role in keeping the ascending duodenum in the abdominal cavity because the duodenum in animals did not belong to a retroperitoneal organ in contrast to humans owing to the difference in the direction of gravity on the abdominal organs between mice and humans. Moreover, the smooth muscle bundle shared common and uncommon points in its location and nerve supply to the suspensory muscle of the duodenum in humans, known as the ligament of Treitz. This study had insufficient evidence that the smooth muscle bundle of the mouse duodenocolic fold was homologous to the suspensory muscle of the duodenum in humans. In conclusion, this study revealed the detailed structure of the mouse duodenocolic fold, including the relationship between the fold and other mesenteries. Particularly, the smooth muscle bundle is a specific feature of the mouse duodenocolic fold and might play several roles in DJF morphogenesis, especially the ascending duodenum and the caudal duodenal flexure during development.

Genetic Influences on the Amount of Cell Death in the Neural Tube of BXD Mice Exposed to Acute Ethanol at Midgestation.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) have a strong genetic component although the genes that underlie this are only beginning to be elucidated. In the present study, one of the most common phenotypes of FASD, cell death within the early developing neural tube, was examined across a genetic reference population in a reverse genetics paradigm with the goal of identifying genetic loci that could influence ethanol (EtOH)-induced apoptosis in the early developing neural tube. METHODS: BXD recombinant inbred mice as well as the parental strains were used to evaluate genetic differences in EtOH-induced cell death after exposure on embryonic day 9.5. Dams were given either 5.8 g/kg EtOH or isocaloric maltose-dextrin in 2 doses via intragastric gavage. Embryos were collected 7 hours after the initial exposure and cell death evaluated via TUNEL staining in the brainstem and forebrain. Genetic loci were evaluated using quantitative trait locus (QTL) analysis at GeneNetwork.org. RESULTS: Significant strain differences were observed in the levels of EtOH-induced cell death that were due to genetic effects and not confounding variables such as differences in developmental maturity or cell death kinetics. Comparisons between the 2 regions of the developing neural tube showed little genetic correlation with the QTL maps exhibiting no overlap. Significant QTLs were found on murine mid-chromosome 4 and mid-chromosome 14 only in the brainstem. Within these chromosomal loci, a number of interesting candidate genes were identified that could mediate this differential sensitivity including Nfia (nuclear factor I/A) and Otx2 (orthodenticle homeobox 2). CONCLUSIONS: These studies demonstrate that the levels of EtOH-induced cell death occur in strain- and region-dependent manners. Novel QTLs on mouse Chr4 and Chr14 were identified that modulate the differential sensitivity to EtOH-induced apoptosis in the embryonic brainstem. The genes underlying these QTLs could identify novel molecular pathways that are critical in this phenotype.

Expression and intracellular localization of Nanos2-homologue protein in primordial germ cells and spermatogonial stem cells.

SummaryThe decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.

Cochleovestibular nerve development is integrated with migratory neural crest cells.

The cochleovestibular (CV) nerve, which connects the inner ear to the brain, is the nerve that enables the senses of hearing and balance. The aim of this study was to document the morphological development of the mouse CV nerve with respect to the two embryonic cells types that produce it, specifically, the otic vesicle-derived progenitors that give rise to neurons, and the neural crest cell (NCC) progenitors that give rise to glia. Otic tissues of mouse embryos carrying NCC lineage reporter transgenes were whole mount immunostained to identify neurons and NCC. Serial optical sections were collected by confocal microscopy and were compiled to render the three dimensional (3D) structure of the developing CV nerve. Spatial organization of the NCC and developing neurons suggest that neuronal and glial populations of the CV nerve develop in tandem from early stages of nerve formation. NCC form a sheath surrounding the CV ganglia and central axons. NCC are also closely associated with neurites projecting peripherally during formation of the vestibular and cochlear nerves. Physical ablation of NCC in chick embryos demonstrates that survival or regeneration of even a few individual NCC from ectopic positions in the hindbrain results in central projection of axons precisely following ectopic pathways made by regenerating NCC.

NANOS2 promotes male germ cell development independent of meiosis suppression.

NANOS2 is an RNA-binding protein essential for fetal male germ cell development. While we have shown that the function of NANOS2 is vital for suppressing meiosis in embryonic XY germ cells, it is still unknown whether NANOS2 plays other roles in the sexual differentiation of male germ cells. In this study, we addressed the issue by generating Nanos2/Stra8 double knockout (dKO) mice, whereby meiosis was prohibited in the double-mutant male germ cells. We found that the expression of male-specific genes, which was decreased in the Nanos2 mutant, was hardly recovered in the dKO embryo, suggesting that NANOS2 plays a role in male gene expression other than suppression of meiosis. To investigate the molecular events that may be controlled by NANOS2, we conducted a series of microarray analyses to search putative targets of NANOS2 that fulfilled 2 criteria: (1) increased expression in the Nanos2 mutant and (2) the mRNA associated with NANOS2. Interestingly, the genes predominantly expressed in undifferentiated primordial germ cells (PGCs) were significantly selected, implying the involvement of NANOS2 in the termination of the characteristics of PGCs. Furthermore, we showed that NANOS2 is required for the maintenance of mitotic quiescence, but not for the initiation of the quiescence in fetal male germ cells. These results suggest that NANOS2 is not merely a suppressor of meiosis, but instead plays pivotal roles in the sexual differentiation of male germ cells.

Proper development of the outer longitudinal smooth muscle of the mouse pylorus requires Nkx2-5 and Gata3.

BACKGROUND & AIMS: Infantile hypertrophic pyloric stenosis is a common birth anomaly characterized by obstruction of the pyloric lumen. A genome-wide association study implicated NKX2-5, which encodes a transcription factor that is expressed in embryonic heart and pylorus, in the pathogenesis of infantile hypertrophic pyloric stenosis. However, the function of the NKX2-5 in pyloric smooth muscle development has not been examined directly. We investigated the pattern of Nkx2-5 during the course of murine pyloric sphincter development and examined coexpression of Nkx2-5 with Gata3 and Sox9-other transcription factors with pyloric-specific mesenchymal expression. We also assessed pyloric sphincter development in mice with disruption of Nkx2-5 or Gata3. METHODS: We used immunofluorescence analysis to compare levels of NKX2-5, GATA3, and SOX9 in different regions of smooth muscle cells. Pyloric development was assessed in mice with conditional or germline deletion of Nkx2-5 or Gata3, respectively. RESULTS: Gata3, Nkx2-5, and Sox9 are coexpressed in differentiating smooth muscle cells of a distinct fascicle of the pyloric outer longitudinal muscle. Expansion of this fascicle coincides with development of the pyloric sphincter. Disruption of Nkx2-5 or Gata3 causes severe hypoplasia of this fascicle and alters pyloric muscle shape. Although expression of Sox9 requires Nkx2-5 and Gata3, there is no apparent hierarchical relationship between Nkx2-5 and Gata3 during pyloric outer longitudinal muscle development. CONCLUSIONS: Nkx2-5 and Gata3 are independently required for the development of a pyloric outer longitudinal muscle fascicle, which is required for pyloric sphincter morphogenesis in mice. These data indicate that regulatory changes that alter Nkx2-5 or Gata3 expression could contribute to pathogenesis of infantile hypertrophic pyloric stenosis.

Transthyretin suppresses the toxicity of oligomers formed by misfolded proteins in vitro.

Although human transthyretin (TTR) is associated with systemic amyloidoses, an anti-amyloidogenic effect that prevents Aß fibril formation in vitro and in animal models has been observed. Here we studied the ability of three different types of TTR, namely human tetramers (hTTR), mouse tetramers (muTTR) and an engineered monomer of the human protein (M-TTR), to suppress the toxicity of oligomers formed by two different amyloidogenic peptides/proteins (HypF-N and Aß42). muTTR is the most stable homotetramer, hTTR can dissociate into partially unfolded monomers, whereas M-TTR maintains a monomeric state. Preformed toxic HypF-N and Aß42 oligomers were incubated in the presence of each TTR then added to cell culture media. hTTR, and to a greater extent M-TTR, were found to protect human neuroblastoma cells and rat primary neurons against oligomer-induced toxicity, whereas muTTR had no protective effect. The thioflavin T assay and site-directed labeling experiments using pyrene ruled out disaggregation and structural reorganization within the discrete oligomers following incubation with TTRs, while confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and hTTR, particularly M-TTR. Moreover, atomic force microscopy (AFM), light scattering and turbidimetry analyses indicated that larger assemblies of oligomers are formed in the presence of M-TTR and, to a lesser extent, with hTTR. Overall, the data suggest a generic capacity of TTR to efficiently neutralize the toxicity of oligomers formed by misfolded proteins and reveal that such neutralization occurs through a mechanism of TTR-mediated assembly of protein oligomers into larger species, with an efficiency that correlates inversely with TTR tetramer stability.

Let-7b and miR-495 stimulate differentiation and prevent metaplasia of pancreatic acinar cells by repressing HNF6.

BACKGROUND & AIMS: Diseases of the exocrine pancreas are often associated with perturbed differentiation of acinar cells. MicroRNAs (miRNAs) regulate pancreas development, yet little is known about their contribution to acinar cell differentiation. We aimed to identify miRNAs that promote and control the maintenance of acinar differentiation. METHODS: We studied mice with pancreas- or acinar-specific inactivation of Dicer (Foxa3-Cre/Dicer(loxP/-) mice), combined (or not) with inactivation of hepatocyte nuclear factor (HNF) 6 (Foxa3-Cre/Dicer(loxP/-)/Hnf6-/- mice). The role of specific miRNAs in acinar differentiation was investigated by transfecting cultured cells with miRNA mimics or inhibitors. Pancreatitis-induced metaplasia was investigated in mice after administration of cerulein. RESULTS: Inhibition of miRNA synthesis in acini by inactivation of Dicer and pancreatitis-induced metaplasia were associated with repression of acinar differentiation and with induction of HNF6 and hepatic genes. The phenotype of Dicer-deficient acini depends on the induction of HNF6; overexpression of this factor in developing acinar cells is sufficient to repress acinar differentiation and to induce hepatic genes. Let-7b and miR-495 repress HNF6 and are expressed in developing acini. Their expression is inhibited in Dicer-deficient acini, as well as in pancreatitis-induced metaplasia. In addition, inhibiting let-7b and miR-495 in acinar cells results in similar effects to those found in Dicer-deficient acini and metaplastic cells, namely induction of HNF6 and hepatic genes and repression of acinar differentiation. CONCLUSIONS: Let-7b, miR-495, and their targets constitute a gene network that is required to establish and maintain pancreatic acinar cell differentiation. Additional studies of this network will increase our understanding of pancreatic diseases.

7,8-Dihydroxyflavone leads to survival of cultured embryonic motoneurons by activating intracellular signaling pathways.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family and a ligand for the tropomyosin-receptor kinase B (TrkB), mediates neuronal survival, differentiation, and synaptic plasticity. However, BDNF is not used to treat neurodegenerative diseases because of its poor pharmacokinetic profile, side effects, and absence of survival properties in clinical trials. Consequently, alternative approaches such as TrkB receptor agonist application are gaining importance. 7,8-Dihydroxyflavone (7,8-DHF), a member of the flavonoid family, has been described as a robust TrkB receptor agonist in hippocampal neurons. Nevertheless, the influence of 7,8-DHF on motoneurons, one of the main targets of BDNF in vivo, is so far unknown. Therefore, we investigated the impact of 7,8-DHF treatment on primary cultured mouse motoneurons. Indeed, we found an activation of the TrkB receptor. Moreover, 7,8-DHF application promotes survival and neurite growth of cultured motoneurons and these effects appear dose-dependent. To investigate the PI3K/AKT and MAPK pathway activation in 7,8-DHF treated motoneurons, we developed a high-density culture system of primary mouse motoneurons. Analysis of both pathways demonstrated a PI3K/AKT but not MAPK pathway activation in cultured motoneurons. This is in contrast to previously published reports about BDNF-mediated activation of TrkB. The lack of MAPK pathway activation is also in contrast to what has been found for hippocampal neurons that indeed show MAPK activation after 7,8-DHF treatment. The ability of 7,8-DHF to imitate BDNF function in motoneurons by using Trk receptor signaling would provide a new approach for the treatment of motoneuron diseases, but needs a more detailed analysis of the activation profile of 7,8-DHF.

The Connexin40A96S mutation from a patient with atrial fibrillation causes decreased atrial conduction velocities and sustained episodes of induced atrial fibrillation in mice.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and a major cause of stroke. In the mammalian heart the gap junction proteins connexin40 (Cx40) and connexin43 (Cx43) are strongly expressed in the atrial myocardium mediating effective propagation of electrical impulses. Different heterozygous mutations in the coding region for Cx40 were identified in patients with AF. We have generated transgenic Cx40A96S mice harboring one of these mutations, the loss-of-function Cx40A96S mutation, as a model for atrial fibrillation. Cx40A96S mice were characterized by immunochemical and electrophysiological analyses. Significantly reduced atrial conduction velocities and strongly prolonged episodes of atrial fibrillation were found after induction in Cx40A96S mice. Analyses of the gating properties of Cx40A96S channels in cultured HeLa cells also revealed significantly lower junctional conductance and enhanced sensitivity voltage gating of Cx40A96S in comparison to Cx40 wild-type gap junctions. This is caused by reduced open probabilities of Cx40A96S gap junction channels, while single channel conductance remained the same. Similar to the corresponding patient, heterozygous Cx40A96S mice revealed normal expression levels and localization of the Cx40 protein. We conclude that heterozygous Cx40A96S mice exhibit prolonged episodes of induced atrial fibrillation and severely reduced atrial conduction velocities similar to the corresponding human patient.

Earliest hematopoietic progenitors at embryonic day 9 preferentially generate B-1 B cells rather than follicular B or marginal zone B cells.

The lymphoid potential of the hematopoietic system is observed as early as embryonic day 9 (E9) before transplantable hematopoietic stem cells (HSCs) appear at E11 in mice. However, it is largely unknown as to which cell fraction is responsible for the initial wave of lymphopoiesis and whether these earliest lymphocytes make any contributions to the adult lymphoid system. We previously isolated the earliest hematolymphoid progenitors at E9 that had CD45(+)c-Kit(+)AA4.1(+) phenotypes. In this study, the differentiation potency into B cell subsets of the E9 hematolymphoid progenitors was examined in detail. In culture, E9 hematolymphoid progenitors produced B220(-/low) B cell progenitors in striking contrast to adult BM c-Kit(+)Sca-1(+)Lin(-) cells. Upon in vivo transplantation, B cell progenitors derived from E9 hematolymphoid progenitors preferentially differentiated into the B-1 B lymphocyte subset, whereas their differentiation into B-2 B lymphocyte subsets [follicular B (FoB), marginal zone B (MZB) cells] was inefficient. Of note, these donor B lymphocytes permanently repopulated in host mice, even if adult mice were used as recipients. These results suggest that B cell progenitors produced from an initial wave of definitive hematopoiesis before authentic HSCs appear could be a permanent source for, at least, the B-1 B lymphocyte subset.

Targeted disruption of fad24, a regulator of adipogenesis, causes pre-implantation embryonic lethality due to the growth defect at the blastocyst stage.

In previous studies, we identified a novel gene, factor for adipocyte differentiation 24 (fad24), which plays an important role during the early stages of adipogenesis in mouse 3T3-L1 cells. Moreover, overexpression of fad24 increased the number of smaller adipocytes in white adipose tissue and improved glucose metabolic activity in mice, thus indicating that fad24 functions as a regulator of adipogenesis in vivo. However, the physiological roles of fad24 in vivo are largely unknown. In this study, we attempted to generate fad24-deficient mice by gene targeting. No fad24-null mutants were recovered after embryonic day 9.5 (E9.5). Although fad24-null embryos were detected in an expected Mendelian ratio of genotypes at E3.5, none of the homozygous mutants developed into blastocysts. In vitro culture experiments revealed that fad24-null embryos develop normally to the morula stage but acquire growth defects during subsequent stages. The number of nuclei decreased in fad24-deficient morulae compared with that in wild-type ones. These results strongly suggested that fad24 is essential for pre-implantation in embryonic development, particularly for the progression to the blastocyst stage.

A short history of hemogenic endothelium.

Definitive hematopoietic cells are generated de novo during ontogeny from a specialized subset of endothelium, the so-called hemogenic endothelium. In this review we give a brief overview of the identification of hemogenic endothelium, explore its links with the HSC lineage, and summarize recent insights into the nature of hemogenic endothelium and the microenvironmental and intrinsic regulators contributing to its transition into blood. Ultimately, a better understanding of the processes controlling the transition of endothelium into blood will advance the generation and expansion of hematopoietic stem cells for therapeutic purposes.

Erythro-myeloid progenitors: "definitive" hematopoiesis in the conceptus prior to the emergence of hematopoietic stem cells.

Erythro-myeloid progenitors (EMP) serve as a major source of hematopoiesis in the developing conceptus prior to the formation of a permanent blood system. In this review, we summarize the current knowledge regarding the emergence, fate, and potential of this hematopoietic stem cell (HSC)-independent wave of hematopoietic progenitors, focusing on the murine embryo as a model system. A better understanding of the temporal and spatial control of hematopoietic emergence in the embryo will ultimately improve our ability to derive hematopoietic stem and progenitor cells from embryonic stem cells and induced pluripotent stem cells to serve therapeutic purposes.

Embryonic hematopoiesis.

Blood cells are continually produced from a pool of progenitors that derive from hematopoietic stem cells (HSCs). In vertebrates, the hematopoietic system develops from two distinct waves or generation of precursors. The first wave occurs in the yolk sac, in mammals or equivalent embryonic structure, and produces nucleated primitive erythrocytes that provide the embryo with the first oxygen transporter and are, therefore, essential for the viability of the embryo. The yolk sac also produces myeloid cells that migrate to the central nervous system and to the skin to form the microglia and skin specific macrophages, the Langerhans cells. The second wave occurs in the dorsal aorta and produces multipotential hematopoietic progenitors. These cells are generated once in the lifetime from mesoderm derivatives closely related to endothelial cells, during a short period of embryonic development. Newly generated cells do not reconstitute the hematopoietic compartment of conventional recipients; therefore, they are designated as immature or pre-HSCs. They undergo maturation into adult HSCs in the aorta or in the fetal liver accompanied by the expression of MHC class I, CD45, CD150, Sca-1 and the absence of CD48. Differentiation of HSCs first occurs in the fetal liver, giving rise to mature blood cells. HSCs also expand in the fetal liver, and in a short time period (four days in the mouse embryo), they increase over 40-fold. HSCs and progenitor cells exit the fetal liver and colonize the spleen, where differentiation to the myeloid lineage and particular lymphoid subsets is favored.

Erythroid development in the mammalian embryo.

Erythropoiesis is the process by which progenitors for red blood cells are produced and terminally differentiate. In all vertebrates, two morphologically distinct erythroid lineages (primitive, embryonic, and definitive, fetal/adult) form successively within the yolk sac, fetal liver, and marrow and are essential for normal development. Red blood cells have evolved highly specialized functions in oxygen transport, defense against oxidation, and vascular remodeling. Here we review key features of the ontogeny of red blood cell development in mammals, highlight similarities and differences revealed by genetic and gene expression profiling studies, and discuss methods for identifying erythroid cells at different stages of development and differentiation.

Regulation of GABA content by glucose in the chick retina.

Some visual information is processed in the retina by γ-aminobutyric acid (GABA) signaling. Once retinal degeneration and visual impairment caused by diabetic retinopathy (DR) are affecting an increasing number of people worldwide, and the disease is characterized by hyper- and hypoglycemic events, the authors aimed to investigate how retinal GABA cell content is affected by variations in glucose availability. Using the ex vivo chick retinas exposed to different glucose concentrations, we observed that amacrine cells from both inner nuclear layer (INL) and ganglion cell layer (GCL) as well as their processes in the inner plexiform layer (IPL) released GABA through GABA transporter-1 (GAT-1) after 30 min of glucose deprivation. Extending this insult to 60 min triggered a permanent loss of GABA-positive amacrine cells, caused swelling of IPL and cell death. High glucose (35 mM) for 30 min induced an increment in GABA immunolabeling in both outer and inner retina. Further, glucose deprivation effects could not be reverted by basal glucose levels and high glucose did not prevent GABA loss upon a glucose deprivation insult. Therefore, GABA cell content is differently affected by short-term variations in glucose availability. While high glucose modulates outer and inner GABAergic circuits, glucose deprivation affects mainly the inner retina. Also, consecutive alteration in glucose supply was not able to rescue basal GABA content. Therefore, glucose oscillations interfering with GABAergic retinal functioning during early stages of retinopathies should be further investigated.

Evaluation of the post-implantation development of mouse embryos derived from round spermatid injection.

Round spermatid injection (ROSI), one of the assistant reproductive technologies, was used to treat partial infertility patients suffering from non-obstructive azoospermia. However, the development efficiency and birth rate of ROSI embryos are extremely low, and it is urgent to investigate the underlying mechanisms of low efficiency to improve the clinical application of ROSI technology. Here, we analyzed and compared the genome stability of the mouse blastocyst and the post-implantation development between ROSI and ICSI embryos. We first sequenced the genome of blastocysts from mouse ROSI embryos that can correctly form male and female pronuclei (2 PN) and found that the genomes of 7 blastocysts were normal. Furthermore, the implantation rate of ROSI 2 PN embryos on embryonic day 7.5 is similar to that of ICSI embryos, and at this time, 37.50% (9/24) of deciduas have no normal gestational sac. The proportion of embryos that survived to embryonic day 11.5 in the ROSI 2 PN group, ROSI non-2 PN group, parthenogenesis group, and ICSI 2 PN group was 51.61%, 7.14%, 0.00%, and 55.00%, respectively. And two smaller fetuses were found in the ROSI 2 PN group, which is not found in the other three groups. In addition, the physiological indexes, including fetus and placenta weight, sex ratio, growth rate, and the natural breeding ability for the offspring obtained from mouse ROSI, were evaluated; ROSI mice exhibited no obvious defect or abnormality and implied that the progeny were safe. Our results provided new evidence to promote the clinical application of ROSI technology.

Administration of retinoic acid to pregnant mice increases the number of fetal mouse glomeruli.

The prevalence of chronic kidney disease (CKD) is increasing worldwide, and CKD is a serious global health problem. Low glomerular number is one of the risk factors for CKD; therefore, the glomerular number is associated with the risk of CKD. Increasing the glomerular number above normal levels may reduce the risk of CKD. It has been reported that, in vitro, the addition of retinoic acid (RA) to the culture medium increases the glomerular number. However, there is no report of an increase in glomerular number above normal levels with the addition of RA in vivo. In this study, RA (20 mg/kg) was administered intraperitoneally to pregnant mice once at embryonic day (E) 10.5, E12.5, E14.5, or E16.5. The fetuses were harvested at E18.5 and fetal mouse kidneys were evaluated. Fetal kidney volume and weight were significantly increased in the E16.5 group compared to the control group. The total glomerular number in the E16.5 group was also approximately 1.46 times higher than that in the control group. In summary, we established a method to increase the glomerular number in the fetal kidney by administration of RA to pregnant mice at E16.5. These results will facilitate the investigation of whether CKD risk is reduced when the glomerular number increases above normal.