Construction of a Mex3c Gene-Deficient Mouse Model to Study C-FOS Expression in Hypothalamic Nuclei and Observe Morphological Differences in Embryonic Neural Tube Development.

BACKGROUND This study utilized CRISPR/Cas9 gene editing technology to construct a Mex3c gene-deficient mouse model, and studied C-FOS expression in hypothalamic nuclei. MATERIAL AND METHODS Thirty Mex3c-/+ mice, 30 mice in the normal group, and 30 Mex3c-/+ mice were randomly divided into control, leptin, and ghrelin groups according to different intraperitoneal injections. HE and Nissl staining were performed to observe the morphology of hypothalamic nerve cells. The C-FOS expression in hypothalamic nuclei of each group was analyzed by immunohistochemical techniques. HE staining was used to observe neural tube morphology, and LFB staining was used to observe nerve myelin sheath morphology. TEM was used to observe neuronal ultrastructure and immunohistochemical techniques were utilized to analyze nestin expression. RESULTS C-FOS expression was lower in the normal control group than in the leptin and ghrelin groups. The Mex3c control group and the leptin group had higher C-FOS expression than the ghrelin group. In neural tube studies, no significant differences were found in the neural tube pathological sections of E14.5-day embryos in each group. Nestin results demonstrated lower expression in the normal group and there was little difference between the HD and Mex3c groups. CONCLUSIONS Mex3c appears to participate in the regulation of energy metabolism by inducing C-FOS expression in the hypothalamus. The neural tubes of the offspring of Mex3c-/+ mice had defects during development.

Temporal candidate gene expression in the sow placenta and embryo during early gestation and effect of maternal Progenos supplementation on embryonic and placental development.

The present study characterised gene expression associated with embryonic muscle development and placental vascularisation during early gestation in the pig and examined effects of Progenos supplementation in early pregnancy. Tissues were collected from commercial multiparous sows (n = 48) from Days 16 to 49 of gestation. In the placenta, qPCR revealed that vascular endothelial growth factor (VEGFA) expression did not change from Day 17 to 49 of gestation; however, KDR receptor and angiopoietin-1 and -2 expression were differentially regulated, with periods of high expression corresponding to two critical phases of angiogenesis in the pig. In the embryo, the pattern of myogenesis-related gene expression was consistent with available literature. A commercially available nutritional supplement Progenos (20 g day⁻¹ L-arginine) added to the diet of sows from either Day 15 to 29 (P15-29; n = 33), Day 30 to 44 (n = 29) or from Day 15 to 44 (n = 76) of gestation tended to increase (P = 0.058) embryonic growth rate compared with non-supplemented controls (n = 79) and angiogenin expression was higher (P = 0.028) at Day 30 of gestation in placentae from sows on the P15-29 Progenos treatment. These results are consistent with proposed beneficial effects of l-arginine on early embryonic development and placental vascularisation.

Precise pattern of recombination in serotonergic and hypothalamic neurons in a Pdx1-cre transgenic mouse line.

BACKGROUND: Multicellular organisms are characterized by a remarkable diversity of morphologically distinct and functionally specialized cell types. Transgenic techniques for the manipulation of gene expression in specific cellular populations are highly useful for elucidating the development and function of these cellular populations. Given notable similarities in developmental gene expression between pancreatic ß-cells and serotonergic neurons, we examined the pattern of Cre-mediated recombination in the nervous system of a widely used mouse line, Pdx1-cre (formal designation, Tg(Ipf1-cre)89.1Dam), in which the expression of Cre recombinase is driven by regulatory elements upstream of the pdx1 (pancreatic-duodenal homeobox 1) gene. METHODS: Single (hemizygous) transgenic mice of the pdx1-creCre/0 genotype were bred to single (hemizygous) transgenic reporter mice (Z/EG and rosa26R lines). Recombination pattern was examined in offspring using whole-mount and sectioned histological preparations at e9.5, e10.5, e11.5, e16.5 and adult developmental stages. RESULTS: In addition to the previously reported pancreatic recombination, recombination in the developing nervous system and inner ear formation was observed. In the central nervous system, we observed a highly specific pattern of recombination in neuronal progenitors in the ventral brainstem and diencephalon. In the rostral brainstem (r1-r2), recombination occurred in newborn serotonergic neurons. In the caudal brainstem, recombination occurred in non-serotonergic cells. In the adult, this resulted in reporter expression in the vast majority of forebrain-projecting serotonergic neurons (located in the dorsal and median raphe nuclei) but in none of the spinal cord-projecting serotonergic neurons of the caudal raphe nuclei. In the adult caudal brainstem, reporter expression was widespread in the inferior olive nucleus. In the adult hypothalamus, recombination was observed in the arcuate nucleus and dorsomedial hypothalamus. Recombination was not observed in any other region of the central nervous system. Neuronal expression of endogenous pdx1 was not observed. CONCLUSIONS: The Pdx1-cre mouse line, and the regulatory elements contained in the corresponding transgene, could be a valuable tool for targeted genetic manipulation of developing forebrain-projecting serotonergic neurons and several other unique neuronal sub-populations. These results suggest that investigators employing this mouse line for studies of pancreatic function should consider the possible contributions of central nervous system effects towards resulting phenotypes.

Prediction of porcine blastocyst formation using morphological, kinetic, and amino acid depletion and appearance criteria determined during the early cleavage of in vitro-produced embryos.

The determination for early cleavage-stage embryos of noninvasive morphologic and metabolic criteria that are predictive of blastocyst development and/or full-term viability remains an important research target. We describe the derivation of a logistic regression model that predicts the probability of porcine blastocyst formation in vitro. Pig zygotes, derived by in vitro maturation and fertilization of slaughterhouse oocytes, were cultured in NCSU-23 medium that was supplemented with a mixture of 20 amino acids (NCSU-23(aa)). On Day 1, at 21, 23, 25, 27, 29 and 31 h postinsemination, cleaving embryos were evaluated morphologically in terms of the: i) number of blastomeres, ii) evenness of division, and iii) degree of fragmentation. These embryos were then placed in 1.5-microl drops of NCSU-23(aa) for 24 h, after which time the three morphologic criteria were re-evaluated and 1.2 microl of spent medium were removed for analysis by HPLC, in order to determine the net rates of amino acid depletion and appearance. Embryos were then cultured singly in NCSU-23(aa) by placing them between the filaments of a woven polyester mesh until Day 6, in order to permit the identification of individual embryos. Of 256 cleaved embryos, 28.7 +/- 6.2% (n = 5 replicates) developed into blastocysts. Discriminant analysis was used to select a subset of amino acids (threonine, valine, lysine, and phenylalanine) that discriminated optimally between embryos that became blastocysts or degenerated. These discriminant scores were entered into the logistic regression. Significant univariate relationships were established between the probability of blastocyst development and amino acid score (odds ratio [OR] 0.53, 95% confidence interval [CI] 0.40-0.69, P < 0.001), cleavage time (OR 0.79, 95% CI 0.71-0.87, P < 0.001), degree of fragmentation on Day 1 (OR 0.55, 95% CI 0.35-0.84, P = 0.009) and Day 2 (OR 0.53, 95% CI 0.35-0.78, P = 0.002), evenness of division on Day 2 (OR 0.66, 95% CI 0.46-0.96, P = 0.028), and categorical values of blastomere number on Day 2 (all P < 0.02), although no single variate could accurately predict blastocyst formation. However, multivariate analysis of the cell numbers on Day 1 and Day 2 correctly classified 51.9% of the predicted blastocysts. The inclusion of cleavage time in the regression analysis raised this rate to 63.5%, which was increased to 66.2% by the addition of evenness of division and degree of fragmentation. Finally, the full logistic regression model, which incorporated amino acid score together with all the other morphologic and kinetic variables, correctly classified 80.8% of the predicted blastocysts. This represented 51.2% of the observed blastocysts. Our data are novel in that they not only define in a quantitative manner the influence of previously undescribed predictors of porcine blastocyst formation, but they also provide a simple model of preimplantation development with reasonable predictive accuracy. The present study also provides a basic model for the examination and incorporation of additional early morphologic and metabolic correlates of developmental competence and could potentially be applied to the selection of human embryos for transfer in clinical IVF.

Ablation of mouse phosphomannose isomerase (Mpi) causes mannose 6-phosphate accumulation, toxicity, and embryonic lethality.

MPI encodes phosphomannose isomerase, which interconverts fructose 6-phosphate and mannose 6-phosphate (Man-6-P), used for glycoconjugate biosynthesis. MPI mutations in humans impair protein glycosylation causing congenital disorder of glycosylation Ib (CDG-Ib), but oral mannose supplements normalize glycosylation. To establish a mannose-responsive mouse model for CDG-Ib, we ablated Mpi and provided dams with mannose to rescue the anticipated defective glycosylation. Surprisingly, although glycosylation was normal, Mpi(-/-) embryos died around E11.5. Mannose supplementation even hastened their death, suggesting that man-nose was toxic. Mpi(-/-) embryos showed growth retardation and placental hyperplasia. More than 90% of Mpi(-/-) embryos failed to form yolk sac vasculature, and 35% failed chorioallantoic fusion. We generated primary embryonic fibroblasts to investigate the mechanisms leading to embryonic lethality and found that mannose caused a concentration- and time-dependent accumulation of Man 6-P in Mpi(-/-) fibroblasts. In parallel, ATP decreased by more than 70% after 24 h compared with Mpi(+/+) controls. In cell lysates, Man-6-P inhibited hexokinase (70%), phosphoglucose isomerase (65%), and glucose-6-phosphate dehydrogenase (85%), but not phosphofructokinase. Incubating intact Mpi(-/-) fibroblasts with 2-[(3)H]deoxyglucose confirmed mannose-dependent hexokinase inhibition. Our results in vitro suggest that mannose toxicity in Mpi(-/-) embryos is caused by Man-6-P accumulation, which inhibits glucose metabolism and depletes intracellular ATP. This was confirmed in E10.5 Mpi(-/-) embryos where Man-6-P increased more than 10 times, and ATP decreased by 50% compared with Mpi(+/+) littermates. Because Mpi ablation is embryonic lethal, a murine CDG-Ib model will require hypomorphic Mpi alleles.

NSCL-1 and NSCL-2 synergistically determine the fate of GnRH-1 neurons and control necdin gene expression.

To study the role of the bHLH genes NSCL-1 and NSCL-2 in the development of GnRH-1 neurons, we have generated compound mutant mice. Mutant animals die at birth and show a virtually complete absence of GnRH-1 neurons in the posterior parts of the brain at E18.5 and an aberrant morphology of the remaining GnRH-1 neurons in the anterior parts of the brain indicating that NSCL-1 and NSCL-2 might concomitantly control differentiation/migration of GnRH-1 neurons in a cell autonomous manner. To gain further insights into this process, we screened for NSCL target genes using DNA array hybridization and detected necdin, which is deleted in the human Prader-Willi syndrome phenotypically resembling the NSCL-2 mutation. Using chromatin immunoprecipitation and site-directed mutagenesis of the necdin promoter, we demonstrate that NSCLs together with additional cofactors directly control transcription of the necdin gene. NSCL-dependent control of necdin expression might be instrumental for proper neuronal cell differentiation and enable GnRH-1 neurons to migrate.

Effect of chemically defined culture medium supplemented with beta-mercaptoethanol and amino acids on implantation and development of different stage in vivo- or in vitro-derived mouse embryos.

In vitro culture (IVC) systems are required for many biotechnological and assisted reproductive technologies and the researchers have been modifying in vitro embryo culture conditions to reach the comparable efficiencies provided in vivo. In the present study, the effects of beta-mercaptoethanol (Beta-ME) and amino acids (AA) on the development of mouse embryos obtained in vivo or in vitro at different stages were investigated. Chemically defined potassium simplex optimized medium (KSOM) was used as basic culture medium and six experimental groups were established and by supplementation of Beta-ME and AA into KSOM media. The quality of blastocysts was evaluated by counting the cells and determining the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells. In addition, embryo transfer (ET) was performed to investigate the rate of implantation and live fetuses. The results obtained in the present study demonstrated that the combined treatment of Beta-ME and AA to 1-cell stage embryos not only enhanced in vitro development to the blastocyst stage but also improved both the number of blastocysts cells and live fetuses.

Identification and characterization of novel developmentally regulated neural-specific proteins, BRINP family.

Processes of neuronal differentiation involve activation of a set of neuronal specific genes and cessation of cell proliferation in postmitotic neurons. Previous studies revealed that bone morphogenetic protein (BMP) and retinoic acid (RA) play important roles in the differentiation of peripheral sympathetic neurons such as the synergistic induction of responsiveness to specific neurotrophic factors. In the present study, while trying to clarify the mechanism of the BMP/RA-actions, we identified a novel neural-specific protein, BMP/RA-inducible neural-specific protein-1 (BRINP1) which shows no similarity to other known proteins. Subsequently, two homologous proteins, BRINP2 and BRINP3, making up the BRINP family, are identified. Individual BRINP genes have distinct regulatory mechanisms of expression within the nervous system. In rodent brain, BRINP1 is expressed from earlier developmental stage, i.e. E9.5, and widely expressed in various neuronal layers and nuclei of the adult animal, while BRINP2 and BRINP3 were detectable from E11.5 and expressed in rather limited regions in a complementary manner. During the course of perinatal development of sympathetic neurons, BRINP1 is induced from earlier embryonic stage and further increased toward adult stage, while BRINP3 expressed from earlier stage is replaced by BRINP2 expression which increases postnatally in accordance with the action of BMP2 and RA. Furthermore, when expressed in nonneuronal cells, all three BRINP family proteins suppressed the cell cycle progression. Possible physiological functions of BRINP family members in the development of the nervous system are discussed.

A study of vehicles for dosing rodent whole embryo culture with non aqueous soluble compounds.

In rodent whole embryo culture (WEC), finding vehicles for non-aqueous-soluble compounds has been problematic due to developmental toxicity associated with many solvents. The purpose of this study was to identify alternative vehicles for insoluble compounds. In WEC, we evaluated carrier solutions containing bovine serum albumin (BSA) and glycerol as well as the solvents, formamide, dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and ethanol, for relative teratogenicity and delivery of the insoluble teratogen, all-trans retinoic acid (RA). At a concentration of

Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens.

Nidogen-1 binds several basement membrane components by well-defined, domain-specific interactions. Organ culture and gene targeting approaches suggest that a high-affinity nidogen-binding site of the laminin gamma1 chain (gamma1III4) is important for kidney development and for nerve guidance. Other proteins may also bind gamma1III4, although human nidogen-2 binds poorly to the mouse laminin gamma1 chain. We therefore characterized recombinant mouse nidogen-2 and its binding to basement membrane proteins and cells. Mouse nidogen-1 and -2 interacted at comparable levels with collagen IV, perlecan, and fibulin-2 and, most notably, also with laminin-1 fragments P1 and gamma1III3-5, which both contain the gamma1III4 module. In embryos, nidogen-2 mRNA was produced by mesenchyme at sites of epithelial-mesenchymal interactions, but the protein was deposited on epithelial basement membranes, as previously shown for nidogen-1. Hence, binding of both nidogens to the epithelial laminin gamma1 chain is dependent on epithelial-mesenchymal interactions. Epidermal growth factor stimulated expression of both nidogens in embryonic submandibular glands. Both nidogens were found in all studied embryonic and adult basement membranes. Nidogen-2 was more adhesive than nidogen-1 for some cell lines and was mainly mediated by alpha3beta1 and alpha6beta1 integrins as shown by antibody inhibition. These findings revealed extensive coregulation of nidogen-1 and -2 expression and much more complementary functions of the two nidogens than previously recognized.

Organization of the mouse dorsal thalamus based on topology, calretinin immnunostaining, and gene expression.

To better understand the organization and evolution of the dorsal thalamus, we have made a first approach to analyze the possible histogenetic compartments of the mammalian dorsal thalamus using mouse embryos. For that, we have analyzed the expression of the proneural gene Math4a and the protein calretinin. Our results suggest the existence of rostrodorsal, caudoventral, and ventral compartments in the embryonic dorsal thalamus of the mouse, which partly parallel the dorsoventral histogenetic tiers postulated in the dorsal thalamus of sauropsids. The rostrodorsal compartment of the mouse dorsal thalamus is characterized by expression of Math4a, and it appears to include sensory and motor thalamic nuclei projecting to the dorsal pallium (isocortex). This compartment appears equivalent to the lemnothalamus proposed by Butler in tetrapods based on hodological grounds. The caudoventral and ventral compartments of the mouse dorsal thalamus lack expression of Math4a in the mantle, but they are characterized by several populations of calretinin-immunorective neurons that show projections to the claustroamygdaloid region in the ventrolateral pallium. More studies will be needed to analyze if the compartments proposed in this study represent true histogenetic units, and to find homologous developmental fields in all vertebrates.

[Metamorphosis of the uterus from Hippocrates to Ambroise Pare]. / Métamorphoses de l'utérus d'Hippocrate à Ambroise Paré.

The treatise Des monstres et prodiges (1579, 1585) by Ambroise Paré includes a vignette depicting a monstrous embryo in the form of a human head surrounded by snakes. This picture belongs to the iconographic tradition relating to the Graeco-Roman mythology of sexuality and procreation. It derives from the belief in the womb's animal nature, illustrated on magic Graeco-Roman and Byzantine gemstones, where the uterus is shown in turn as a cupping vessel, a scarab-beetle, an octopus or the head of Gorgo.

In vivo requirements for GATA-1 functional domains during primitive and definitive erythropoiesis.

GATA-1 is a transcription factor essential for erythroid/megakaryocytic cell differentiation. To investigate the contribution of individual domains of GATA-1 to its activity, transgenic mice expressing either an N-terminus, or an N- or C-terminal zinc finger deletion of GATA-1 (Delta NT, Delta NF or Delta CF, respectively) were generated and crossed to GATA-1 germline mutant (GATA-1.05) mice. Since the GATA-1 gene is located on the X-chromosome, male GATA-1 mutants die by embryonic day 12.5. Both Delta NF and Delta CF transgenes failed to rescue the GATA-1.05/Y pups. However, transgenic mice expressing Delta NT, but not the Delta NF protein, were able to rescue definitive hematopoiesis. In embryos, while neither the Delta CF protein nor a mutant missing both N-terminal domains (Delta NTNF) was able to support primitive erythropoiesis, the two independent Delta NT and Delta NF mutants could support primitive erythropoiesis. Thus, lineage-specific transgenic rescue of the GATA-1 mutant mouse revealed novel properties that are conferred by specific domains of GATA-1 during primitive and definitive erythropoiesis, and demonstrate that the NT and NF moieties lend complementary, but distinguishable properties to the function of GATA-1.

Spatio-temporally regulated expression of receptor tyrosine kinases, mRor1, mRor2, during mouse development: implications in development and function of the nervous system.

BACKGROUND: Drosophila neurospecific receptor tyrosine kinases (RTKs), Dror and Dnrk, as well as Ror1 and Ror2 RTKs, isolated from human neuroblastoma, have been identified as a structurally related novel family of RTKs (Ror-family RTKs). Thus far, little is known about the expression and function of mammalian Ror-family RTKs. RESULTS: We have identified murine Ror-family RTKs, mRor1 and mRor2. Both mRor1 and mRor2 genes are induced upon neuronal differentiation of P19EC cells. During neuronal differentiation in vitro, the expression of mRor2 is transiently induced, although that of mRor1 increases continuously. During embryogenesis, the mRor1 gene is expressed in the developing nervous system within restricted regions and in the developing lens epithelium. The expression of mRor1 is sustained in the nervous system and is also detected in non-neuronal tissues after birth. In contrast, the expression of mRor2 is detected mainly in the developing nervous system within broader regions and declines after birth. Possible relationships of mRor1 and mRor2 genes with previously identified mutants have also been examined. CONCLUSIONS: The developmental expressions of mRor1 and mRor2, in particular in the nervous system, are differentially regulated, reflecting their expression patterns in vitro. mRor1 and mRor2 may thus play differential roles during the development of the nervous system.

Cyclin-dependent kinase 5-deficient mice demonstrate novel developmental arrest in cerebral cortex.

The cerebral cortex of mice with a targeted disruption in the gene for cyclin-dependent kinase 5 (cdk5) is abnormal in its structure. Bromodeoxyuridine labeling reveals that the normal inside-out neurogenic gradient is inverted in the mutants; earlier born neurons are most often found superficial to those born later. Despite this, the early preplate layer separates correctly and neurons with a normal, pyramidal morphology can be found between true marginal zone and subplate. Consistent with their identity as layer VI corticothalamic neurons, they can be labeled by DiI injections into thalamus. The DiI injections also reveal that the trajectories of the cdk5(-/-) thalamocortical axons are oblique and cut across the entire cortical plate, instead of being oriented tangentially in the subcortical white matter. We propose a model in which the cdk5(-/-) defect blocks cortical development at a heretofore undescribed intermediate stage, after the splitting of the preplate, but before the migration of the full complement of cortical neurons.

Effects of surgical decapitation and chicken growth hormone (cGH) replacement therapy on chick embryo growth.

The effects of decapitation and chicken growth hormone (cGH) replacement therapy on chick embryo growth has been investigated. Removal of the prosencephalon at 33-38 hrs (1.38 to 1.58 Days) of incubation decreased body (torso) and liver weights as well as skeletal growth as indicated by tibial length. A single pituitary gland transplanted onto the chorioallantoic membrane (CAM) partially restored torso growth and completely reversed the increase in body water content which characterizes decapitated embryos. Replacement therapy with cGH did not influence body weight but did, on Day 16.5 of incubation, increase tibial length and liver DNA content and concentration. These latter findings suggest that there may be limited hypothalamoadenohypophyseal (GH) axis function in the chick embryo. The effects of decapitation on torso growth are also discussed in conjunction with decapitation effects on albumen swallowing (absorption) and yolk absorption.

A new method to express embryotoxic data obtained in vitro on whole murine embryos.

This report proposes to express the effect of drugs in the whole embryo culture system by a new method using an intrinsic reference. Percentages of malformed embryos or other defects in development, expressed as percentages of controls, are plotted against concentrations of the drug expressed as percentages of the concentration inducing 50% embryolethality (ELC50). It is suggested that the slope generated by this method is directly related to the intensity of the in vitro teratogenic potential of the agent and allows an estimation of the specific interference with developmental processes. The method has been applied to data obtained from the literature and pertaining to 22 drugs. The slope generated by these drugs varied widely. The effect of these drugs could be meaningfully compared in spite of the wide range of ELC50 displayed by the drugs. In addition, results obtained for two drugs in different laboratories using different methods and species were in fair agreement when they were compared by using the proposed method. Finally, it is suggested that the method provides an improved means to inquire if there is any relevance of in vitro data to teratological results obtained in vivo.