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PubMed2006

Genetic interaction between Lef1 and Alx4 is required for early embryonic development.

Lymphoid Enhancer Factor-1 (Lef1) facilitates the assembly of transcriptional regulatory complexes and mediates nuclear responses to Wnt signals. We determined previously that the mesenchymally restricted, paired-like homeodomain protein Aristaless-like 4 (Alx4) interacts with Lef1 and together alters promoter activity of candidate genes. In order to define their overlapping functions, mice deficient for both Lef1 and Alx4 activity (Lef1-/-/Alx4lstD/lstD) were produced. Whereas embryos lacking either Lef1 or Alx4 activity remain viable up to or after birth, early embryonic lethality results when both factors were absent. No viable Lef1-/-/Alx4lstD/lstD embryos were recovered beyond 9.5 dpc. Between E8.5 and E10, viable Lef1-/-/Alx4lstD/lstD embryos were developmentally delayed 0.5 days relative to littermates of all other genotypes. Principle among the alterations seen in Lef1-/-/Alx4lstD/lstD animals was defective vasculature in both embryonic and extra-embryonic tissues. In the yolk sac, while the vascular network is present, it were greatly diminished and large vitelline vessels were largely absent. Platelet/endothelial cell adhesion molecule (PECAM) staining revealed that the major vessels in the head of compound mutant embryos were absent, while the other vessels were finer than those seen in normal littermates. Pools of blood and pericardial effusion were also apparent in Lef1-/-/Alx4lstD/lstD animals, further indicative of a defective vasculature. These data confirm genetically the interaction between Lef1 and Alx4 and further reveal unknown, overlapping roles for these transcription factors in embryonic vasculogenesis.

Animals↗

PubMed2006

Developmental consequences of sexual dimorphism during pre-implantation embryonic development.

Abnormalities of development potential arising from pre-implantation environment are not limited to in vitro culture (IVC) (for, i.e. in ruminants the large offspring syndrome produced by IVC), they may also be consequence of specific stress conditions experienced in vivo, like maternal diet, toxins, etc. A complex group of mechanisms (gene expression, epigenetic, metabolic, etc.) may operate to link early embryo environment with future health. Furthermore, during the pre-implantation period, in vitro produced male embryos have a higher metabolic rate, they grow faster than females, and they also have differential gene transcription of genes located in the Y-, X-, or in autosomal-chromosomes. As a consequence of these differences embryos may be affected differentially by natural or artificial environmental conditions, depending on their gender. It has been suggested that under some stress conditions male embryos are more vulnerable than females; however the biological fragility of male embryos is poorly understood. Evidences suggest that epigenetic differences produced by the presence of one or two X-chromosomes are the principal cause of the male and female pre-implantation differences, and we put forward the possible role of these early sex differences to control sex ratio of the offspring under different environmental conditions in Nature. By following the differences between male and female early embryos not only may be possible to manipulate sex ratio in farm animals, we can also gain further insight into aspects of early embryo development, X inactivation, and epigenetic and genetic processes related with early development that may have a long-term effect on the offspring.

Animals↗

PubMed1986

Increased mortality during early embryonic development after in-vitro fertilization of rat oocytes.

Immature female rats (60-65 g) were injected with 4 i.u. PMSG on Day -2, and allocated to 3 groups. For Groups I and II, unmated donors were killed 67-69 h after PMSG injection, shortly after the expected time of ovulation. Oocytes were recovered from the oviducts and transferred immediately into the oviduct of mated recipients (Group I) whose ipsilateral ovary had been exposed by peeling back the bursa, preventing endogenous oocytes from entering the oviduct, or were fertilized in vitro (Group II) and were transferred 16-18 h later. Rats in Group III were allowed to mate and half were killed 6 h after mating. The fertilized oocytes were then incubated for 10-12 h until transfer. The remaining rats in Group III were killed 16-18 h after mating and fertilized oocytes were collected and transferred immediately. Recipient rats were killed on Days 2, 5, 8 and 20. Zygotes resulting from in-vitro fertilization (Group II) were as able as those fertilized in donors (Group III) or recipients (Group I) to develop to the 2-cell stage, but underwent significantly greater embryonic loss beyond this stage of development. There was a slower rate of development of such oocytes to the blastocyst stage (Day 5) and a lower mean weight of implantation sites (Day 8). Transfer of zygotes after in-vitro fertilization resulted in a loss of 35% of the embryos at the time of implantation. These results suggest that in-vitro fertilization of rat oocytes leads to defects in the embryos causing a delay in early embryo development and a large number of implantation losses.

Animals↗

PubMed2005

Nuclear translocation of phospholipase C-zeta, an egg-activating factor, during early embryonic development.

Phospholipase C-zeta (PLCzeta), a strong candidate of the egg-activating sperm factor, causes intracellular Ca2+ oscillations and egg activation, and is subsequently accumulated into the pronucleus (PN), when expressed in mouse eggs by injection of RNA encoding PLCzeta. Changes in the localization of expressed PLCzeta were investigated by tagging with a fluorescent protein. PLCzeta began to translocate into the PN formed at 5-6 h after RNA injection and increased there. Observation in the same embryo revealed that PLCzeta in the PN dispersed to the cytoplasm upon nuclear envelope breakdown and translocated again into the nucleus after cleavage. The dynamics was found in the second mitosis as well. When RNA was injected into fertilization-originated 1-cell embryos or blastomere(s) of 2-8-cell embryos, the nuclear localization of expressed PLCzeta was recognized in every embryo up to blastocyst. Thus, PLCzeta exhibited alternative cytoplasm/nucleus localization during development. This supports the view that the sperm factor could control cell cycle-dependent generation of Ca2+ oscillations in early embryogenesis.

Active Transport, Cell Nucleus↗

PubMed2004

Impact of oxygen concentration on embryonic development of mouse zygotes.

The aim of the present study was to examine the effect of culture under 5 and 20% oxygen on the development, differentiation and viability of zygotes and in-vivo-produced embryos at the 2-cell and 8-cell stages of development. First, zygotes collected in a common pool were cultured in 20% O2 for 0, 23, 46 and 95 h. Zygotes and in-vivo-produced embryos at the 2-cell and 8-cell stages of development were then cultured in 5 or 20% O2. The proportion of embryos reaching the compaction and blastocyst stages of development did not differ between groups regardless of the period of time embryos were cultured in 20% O2 or the stage at beginning of culture. Duration of culture under 20% O2 had a significant effect on total number of blastocyst cells. A stage-specific effect was observed on total and trophectoderm cell numbers in blastocysts resulting from the culture of zygotes and in-vivo-produced embryos under 20% O2. ICM and percent ICM development was significantly decreased by culture in 20% O2 at all stages examined. Oxygen concentration had no effect on implantation rate and fetal weights upon embryo transfer. However, transfer of zygotes grown to the blastocyst stage in 20% O2 resulted in a dramatic decrease in fetal development per blastocyst and fetal development per implantation. These results demonstrate that culture of F1 mouse zygotes in 20% O2 compromises the developmental potential of resultant blastocysts, which appear to be normal on morphological assessment.

Animals↗

PubMed2006

Hepatocyte nuclear factor 4alpha is essential for embryonic development of the mouse colon.

BACKGROUND & AIMS: Hepatocyte nuclear factor 4 alpha (HNF4alpha) is a transcription factor that has been shown to be required for hepatocyte differentiation and development of the liver. It has also been implicated in regulating expression of genes that act in the epithelium of the lower gastrointestinal tract. This implied that HNF4alpha might be required for development of the gut. METHODS: Mouse embryos were generated in which Hnf4a was ablated in the epithelial cells of the fetal colon by using Cre-loxP technology. Embryos were examined by using a combination of histology, immunohistochemistry, DNA microarray, reverse-transcription polymerase chain reaction, electrophoretic mobility shift assays, and chromatin immunoprecipitation analyses to define the consequences of loss of HNF4alpha on colon development. RESULTS: Embryos were recovered at E18.5 that lacked HNF4alpha in their colons. Although early stages of colonic development occurred, HNF4alpha-null colons failed to form normal crypts. In addition, goblet-cell maturation was perturbed and expression of an array of genes that encode proteins with diverse roles in colon function was disrupted. Several genes whose expression in the colon was dependent on HNF4alpha contained HNF4alpha-binding sites within putative transcriptional regulatory regions and a subset of these sites were occupied by HNF4alpha in vivo. CONCLUSIONS: HNF4alpha is a transcription factor that is essential for development of the mammalian colon, regulates goblet-cell maturation, and is required for expression of genes that control normal colon function and epithelial cell differentiation.

Animals↗

PubMed1997

Gene expression and immunolocalisation of amelogenins in developing embryonic and neonatal hamster teeth.

Amelogenins are a group of related matrix proteins, synthesised and secreted by ameloblasts during the formation of dental enamel. We have examined expression patterns and the tissue distribution of amelogenins by in situ hybridisation and by immunohistochemistry of developing teeth of embryonic (E12-E15) and neonatal (1- to 4-day-old) golden hamsters. Amelogenin expression and (intracellular) immunostaining for amelogenins were first observed in late embryonic stages in E14 incisors and E15 first molars in partially polarised pre-ameloblasts located along a thin layer of predentine before any overt deposition of enamel. Expression of mRNA and protein staining for amelogenins increased with age and early pre-dentine became immunopositive. The highest mRNA levels and substantial immunostaining for amelogenins were noted in neonatal-stage secretory ameloblasts fully engaged in enamel matrix deposition. After completion of the secretory phase, amelogenin gene expression continued at a lower level in post-secretory stages and was seen in transition-phase and maturation-phase ameloblasts. No amelogenin transcripts were observed in odontoblasts at any stage of their development. However, young odontoblasts stained weakly with anti-amelogenin antibodies before they formed the first layer of dentine, although this staining disappeared in odontoblasts at later stages of development. We conclude that amelogenin gene transcription occurs as early as the polarisation stage of pre-ameloblasts and is closely followed by translation of mRNA into amelogenin proteins. Odontoblasts do not transcribe the amelogenin gene and probably endocytose and digest amelogenins from the pre-dentine. Amelogenins are also transcribed but at a low level in post-secretory stages of amelogenesis.

Aging↗

PubMed1975

[Embryonic and post-embryonic development of the parietal and visceral peritoneum in white mice].

Light and electron microscopy were used in order to investigate histogenesis of the parietal and visceral peritoneum of white mice in embryonic and postembryonic periods of development. Four periods were distinguished, during which gradual differentiation of the primordium material into tissue structures (mesothelium and the connective tissue) of the peritoneum were observed. Asynchronous differentiation of the mesothelium as well as certain correlation in the degree of differentiation of mesothelial and mesenchymal cells took place at all stages of the embryonic and postembryonic development. More differentiated cells of prolonged shape were predominant in the mesenchyma even at early stages (11 days) in those portions where the lining of the secondary cavity of the body resembled mesothelim in its structure.

Animals↗

PubMed1999

Mechanism of neurogenesis during the embryonic development of a tunicate.

Ascidian and vertebrate nervous systems share basic characteristics, such as their origin from a neural plate, a tripartite regionalization of the brain, and the expression of similar genes during development. In ascidians, the larval chordate-like nervous system regresses during metamorphosis, and the adult's neural complex, composed of the cerebral ganglion and the associated neural gland is formed. Classically, the homology of the neural gland with the vertebrate hypophysis has long been debated. We show that in the colonial ascidian Botryllus schlosseri, the primordium of the neural complex consists of the ectodermal neurohypophysial duct, which forms from the left side of the anterior end of the embryonal neural tube. The duct contacts and fuses with the ciliated duct rudiment, a pharyngeal dorsal evagination whose cells exhibit ectodermic markers being covered by a tunic. The neurohypophysial duct then differentiates into the neural gland rudiment whereas its ventral wall begins to proliferate pioneer nerve cells which migrate and converge to make up the cerebral ganglion. The most posterior part of the neural gland differentiates into the dorsal organ, homologous to the dorsal strand. Neurogenetic mechanisms in embryogenesis and vegetative reproduction of B. schlosseri are compared, and the possible homology of the neurohypophysial duct with the olfactory/adenohypophysial/hypothalamic placodes of vertebrates is discussed. In particular, the evidence that neurohypophysial duct cells are able to delaminate and migrate as neuronal cells suggests that the common ancestor of all chordates possessed the precursor of vertebrate neural crest/placode cells.

Animals↗

PubMed2005

Successful pig embryonic development in vitro outside a CO2 gas-regulated incubator: effects of pH and osmolality.

We investigated the effects of HEPES in the medium (to maintain pH) and paraffin oil covering the medium (to maintain osmolality) on the developmental ability of porcine embryos produced in vitro using tightly closed glass tubes in the absence of a CO2 gas-regulated incubator. Putative porcine zygotes obtained by in vitro fertilization (IVF) of in vitro-matured (IVM) oocytes (day of IVF=Day 0) were cultured in 5% CO2 gas-equilibrated NCSU-37 media containing pyruvate and lactate during Days 0-2, and glucose during Days 2-6, in open glass tubes in a CO2 incubator or tightly closed glass tubes without a CO2 incubator at 38.5 degrees C. The following four media were used: (1) medium covered with paraffin oil and supplemented with HEPES; (2) medium covered with paraffin oil but with no HEPES supplementation; (3) medium not covered with paraffin oil but supplemented with HEPES; (4) medium not covered with paraffin oil and with no HEPES supplementation. As a control group, zygotes were cultured in medium with neither paraffin oil coverage nor HEPES supplementation using a four-well dish in a CO2 gas-regulated incubator. After culture, the osmolality in each of the four closed conditions was maintained at approximately 285-286 mOsm, lower (P<0.05) than that in the control (291 mOsm). In the two HEPES-supplemented media groups in the closed-tube system, the pH was maintained at 7.5-7.7, and the blastocyst development rates (15.5% in non-oil covered and 18.5% in oil covered group) did not differ significantly from that of the control (20.2%), although the mean cell numbers in the blastocysts in the two closed-tube condition groups (28.2 and 33.0) were lower (P<0.05) than in the control (43.5). In contrast, the pH was higher in the two groups without HEPES supplementation (approximately 8.0) than the control (7.4; P<0.05), and the blastocyst development rates (10.9% in non-oil covered and 7.5% in oil covered group) or total cell numbers in the blastocyst (24.8 and 28.7) in the two non-HEPES groups were drastically decreased (P<0.05) compared to those in the control (20.2% and 43.5). These results suggested that maintenance of pH is important for successful in vitro porcine embryo culture under closed-air conditions, whereas the range of osmolality that suits embryo development is not limited to a small range. Furthermore, blastocyst production was possible in a glass tube without a CO2 incubator, although blastocyst quality was lower compared to those produced in an incubator.

Animals↗

PubMed2006

Gene profiling and bioinformatic analysis of Schwann cell embryonic development and myelination.

To elucidate the molecular mechanisms involved in Schwann cell development, we profiled gene expression in the developing and injured rat sciatic nerve. The genes that showed significant changes in expression in developing and dedifferentiated nerve were validated with RT-PCR, in situ hybridisation, Western blot and immunofluorescence. A comprehensive approach to annotating micro-array probes and their associated transcripts was performed using Biopendium, a database of sequence and structural annotation. This approach significantly increased the number of genes for which a functional insight could be found. The analysis implicates agrin and two members of the collapsin response-mediated protein (CRMP) family in the switch from precursors to Schwann cells, and synuclein-1 and alphaB-crystallin in peripheral nerve myelination. We also identified a group of genes typically related to chondrogenesis and cartilage/bone development, including type II collagen, that were expressed in a manner similar to that of myelin-associated genes. The comprehensive function annotation also identified, among the genes regulated during nerve development or after nerve injury, proteins belonging to high-interest families, such as cytokines and kinases, and should therefore provide a uniquely valuable resource for future research.

Agrin↗

PubMed1989

Correlation of embryonic development and adult neoplastic changes of human prostate.

We have studied embryonic and fetal differentiation of the human prostate in relation to androgen-producing Leydig cell differentiation. We have studied the differentiation of human prostatic glands and the synthesis of acid phosphatase in vivo and in vitro. These studies have shown that the mesenchyme at the level of the openings of the para- and mesonephric ducts to the urethra was the local initiator of prostatic differentiation. All prostatic acini developed by epithelial outgrowths from the urethral epithelium. None of them grew from para- or mesonephric ducts. However, the epithelium on the colliculus seminalis differed from the rest of the urethral epithelium morphologically and in acid phosphatase content. Androgens accelerated differentiation in vitro and acid phosphatase activity was shown to be present in prostatic urethral epithelium and prostatic acini both in vivo and in vitro. According to these studies embryonic differentiation gives no direct answer to the localisation of adult neoplastic changes in different parts of the prostate, although in the posterior part there might be a mixture of cells from ductal and urethral epithelium. Secretion of acid phosphatase seems to be a constitutional phenomenon of this part of epithelium and is partly regulated by androgens. Epitheliomesenchymal interaction is important in differentiation and the role of this interaction in adult diseases might be valuable to be studied.

Acid Phosphatase↗

PubMed2006

In vivo study of apoptosis as a creative agent of embryonic development of the primary nasal duct in rats.

INTRODUCTION: The first embryonic part of the nasal cavity is the primary nasal duct, beginning with the olfactory placode and ending with the oronasal membrane. Aim of this study was to investigate the cellular processes (apoptosis, proliferation) being responsible for development and opening of the primary nasal duct. MATERIAL AND METHODS: In this study developmental processes in at least three regions of the primary nasal duct (opening, middle, end) were examined by sectioning 38 rat fetuses on day 13.5 after conception. Apoptotic cells were detected by active caspase-3 antibodies and proliferating cells were examined by Ki-67 antibodies. RESULTS: Multiple apoptotic events were diagnosed on the basis and proliferative cells on the top of this duct. CONCLUSION: Apoptosis and proliferation play an important role in the process of opening the bottom of the primary nasal duct and for development of the nasal septum, philtrum as well as the primary palate. Mesenchymal proliferation seems to play a minor role in the process of opening the primary nasal duct.

Animals↗

PubMed2005

Expression profile of the RNA-binding protein gene hermes during chicken embryonic development.

The hermes gene encodes an RNA-binding protein containing an RNA-recognition motif. Its expression has been described previously in Xenopus and in the developing heart of very young chicken embryos. We have analyzed the expression of cHermes in later heart development, where expression is maintained in the myocardium, and also in previously undescribed sites. cHermes expression first appears in the somites in the first terminally differentiated myocytes of both the epaxial and the hypaxial myotome. Expression is also seen in the primordium of the allantois and continues in the developing allantoic sac. cHermes expression in the pronephric and mesonephric kidneys coincides temporally and spatially with the appearance of the vascular components of the glomeruli. In addition, cHermes expression was seen in the mesoderm of the gut and in the notochord.

Allantois↗

PubMed2006

Maternal serotonin is crucial for murine embryonic development.

The early appearance of serotonin and its receptors during prenatal development, together with the many effects serotonin exerts during CNS morphogenesis, strongly suggest that serotonin influences the development and maturation of the mammalian brain before it becomes a neuromodulator/neurotransmitter. Sites of early serotonin biosynthesis, however, have not been detected in mouse embryos or extraembryonic structures, suggesting that the main source of serotonin could be of maternal origin. This hypothesis was tested by using knockout mice lacking the tph1 gene, which is responsible for the synthesis of peripheral serotonin. Genetic crosses were performed to compare the phenotype of pups born from homozygous and heterozygous mothers. Observations provide the first clear evidence that (i) maternal serotonin is involved in the control of morphogenesis during developmental stages that precede the appearance of serotonergic neurons and (ii) serotonin is critical for normal murine development. Most strikingly, the phenotype of tph1-/- embryos depends more on the maternal genotype than on that of the concepti. Consideration of the maternal genotype may thus help to clarify the influence of other genes in complex diseases, such as mental illness.

Animals↗

PubMed1991

Embryonic development and survival of brachial motoneurons projecting to muscleless chick wings.

The role of muscle cells in the survival of embryonic motoneurons projecting to the developing wing was directly examined. Embryos lacking muscle in one of their wings were produced by surgically removing the embryonic precursors of muscle cells, the somites. The resulting limb lacked only muscle cells, with the derivatives of the other limb contributor, the lateral plate mesoderm, left intact. Counts of apparently healthy lateral motor column (LMC) motoneurons supplying normal wings between stages 28 and 36 showed little decline in motoneuron number until stage 34; approximately 24% of the motoneurons died between stages 34 and 36. In contrast, the number of LMC motoneurons supplying muscleless wings declined progressively from stages 28 to 36. This decline resulted in the loss of about 77% of the motoneurons present at stage 28. In addition, the LMCs supplying muscleless wings had fewer motoneurons at all stages examined than similarly staged controls; this difference ranged from 27% in the youngest cases to 75% in the older embryos. Motoneurons were lost equivalently from all rostrocaudal levels of the brachial LMC. From these studies we conclude that motoneurons survival depends on the presence of muscle cells in the developing wing. In the absence of muscle cells, motoneuron death was increased compared to normal embryos at stages prior to the onset of naturally occurring cell death.

Animals↗

PubMed2005

A proteomic analysis of mammalian preimplantation embryonic development.

Genetic studies on the mammalian preimplantation embryo are providing a wealth of information regarding gene expression. However, changes in the transcriptome do not always reflect cellular function or the complexity and diversity of the mammalian proteome with post-translational modifications or protein-protein interactions. To elucidate embryonic cellular function, a detailed understanding at the protein level is necessary. The aim of this study was to generate protein profiles of mammalian embryos throughout development, and to investigate the effects of oxygen concentration on the embryonic proteome. A protocol was developed to analyse small groups of embryos (n = 5) by time-of-flight mass spectrometry. F1 mice zygotes were cultured in G1/G2 sequential media with recombinant albumin (2.5 mg/ml) in 6% CO(2) and O(2) concentrations of either 5% or 20%. In vivo-developed embryos were flushed from the reproductive tract (day 4). Protein profiles were generated for all embryonic samples and statistical analysis revealed 32 potential proteins/biomarkers with significant changes (P < 0.05). Embryos generated under 5% O(2) more closely resembled in vivo-developed embryos. Under 20% O(2) conditions, embryos showed down-regulation of 10 proteins/biomarkers (masses between 4 to 20 kDa) (P < 0.05) confirming the pathological effects of oxygen during embryonic development. These data demonstrate for the first time the complexity of the mammalian preimplantation proteome. The unique protein profiles of in vivo-developed embryos and a panel of selected biomarkers represent optimal cellular function, against which comparisons can be made to facilitate improvements in mammalian assisted reproduction techniques procedures.

Animals↗

PubMed1993

Messenger RNA phenotyping for semi-quantitative comparison of glucocorticoid receptor transcript levels in the developing embryonic mouse palate.

A specific complex of glucocorticoid and receptor interacts with responsive genes in target tissues of developing palate and controls its morphogenesis. In order to examine developmental changes in glucocorticoid receptor gene expression during embryonic palate formation, variations in transcript levels have been determined using a simple semi-quantitative reverse transcription-polymerase chain reaction (PCR) procedure. Oligo (dT)16 was used as the primer for reverse transcription, and the amount of PCR product for glucocorticoid receptor was normalized against those for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin. We found that either GAPDH or beta-actin were suitable internal standards, and that the mRNA levels of glucocorticoid receptor undergoes a significant decrease of 30% at the developmental stage of embryonic mouse palatal shelf re-orientation. Immunolocalization of the receptor demonstrated a significant change in the distribution pattern of glucocorticoid receptor during the re-orientation stage of secondary palate formation. Our present data suggest that developmental variations in glucocorticoid receptor level control morphogenetic events of secondary palate formation.

Animals↗