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PubMed1992

Expression of estrogen receptor gene in mouse oocyte and during embryogenesis.

Estrogen is required for oocyte maturation and embryonic development in vivo; however, the mechanism involved is not clear. Since the effect of estrogen is mediated through the estrogen receptor (ER), we examined the ontogeny and expression of the ER gene in mouse oocytes and embryos of various gestational stages using the highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Total RNA, extracted from 40 ovulated oocytes, 2-cell embryos, morulae, and blastocysts, was reverse transcribed into cDNA. A pair of primers flanking the 453-bp region encoding the hormone-binding domain of ER was used for 30 cycles of PCR. The identity of the amplified product was confirmed by sizing and Southern blot hybridization. The results indicated that ER gene is expressed in unfertilized oocytes and cumulus-oocyte complexes. The amount of ER mRNA decreases in 2-cell embryos, coincident with degradation of maternal mRNA. No ER transcript can be detected in the morulae or blastocyst stage when the embryonic genome has been activated. Postimplantation embryos do not contain detectable ER mRNA until gestation day 8. The levels of ER mRNA increase from day 10 to day 18 of gestation. These data suggest that estrogen, secreted by granulosa cells, may directly influence oocyte growth and maturation in vivo. Since estrogen is known to stimulate the production of growth factors in mouse uteri, the absence of ER mRNA in periimplantation embryos suggests that the effects of estrogen on early embryogenesis may be indirect, i.e., through estrogen-regulated growth-promoting factors produced by the reproductive tract. In mid- and late-post-implantation embryos which contain ER mRNA, estrogen may affect embryonic development through the receptor-mediated mechanisms.

Animals↗

PubMed1994

Fate of sperm organelles during early embryogenesis in the rat.

This report is part of a continuing study in which we employ monoclonal antibodies to membrane domains and internal organelles of rat spermatozoa in order to trace events during maturation, capacitation, fertilization, and early development. In the present study, we have used immunocytochemistry at the light and EM levels to localize one antibody, 5A5, to the fibrous sheath and a second, 3D5, to the outer mitochondrial membrane. Antibody 5A5 does not stain the fibrous sheath of spermatozoa of rodents other than the rat, while 3D5 can be localized to the outer mitochondrial membrane of rat, hamster, and mouse spermatozoa. In order to follow these antibodies during fertilization and early embryogenesis, we developed a method to stain internal components of zygotes and early embryos. Our findings suggests that the fibrous sheath disappears prior to the first cleavage and that mitochondria can be detected up to the 2-cell stage in mouse and the 4-cell stage in rat.

Animals↗

PubMed2006

Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, beta-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitro.

Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, measurement of gene expression generally requires selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic development, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have validated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and histone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed acceptable rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycerokinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development.

Actins↗

PubMed2001

Monitoring the production yields of vincristine and vinblastine in Catharanthus roseus from somatic embryogenesis. Semiquantitative determination by flow-injection electrospray ionization mass spectrometry.

A semiquantitative determination of two bis-indole antitumor alkaloids, vincristine and vinblastine, has been performed by flow-injection electrospray ionization mass spectrometric analysis of the extracts of Catharanthus roseus. Leaves and flowers of two different phenotypes (pink flower and white flower) obtained from somatic embryogenesis were thus examined and compared with the field-grown mother plant. Different amounts of vincristine and vinblastine were detected depending on the examined samples.

Antineoplastic Agents, Phytogenic↗

PubMed1985

pH-induced changes in mitotic and developmental patterns in sea urchin embryogenesis. I. Exposure of embryos.

The effects of different pH conditions have been investigated on sea urchin larval development following exposure of embryos to controlled, though changing, decreases or increases of seawater pH. The pH of filtered natural seawater was initially adjusted with 1 N HCl of 1 N KOH and then was altered back to its normal values (8.0-8.2) by the exchange with atmospheric CO2 and subsequent carbonic acid equilibrium. During cultures, pH was regularly monitored. When developing embryos were reared in different pH conditions, larval differentiation was sharply affected by an apparently moderate pH decrease, such as 0.5 pH units. However, even pH decreases as small as 0.2 pH units from the normal value showed reproducible damage to embryogenesis. This damage appeared to be early and irreversible, since the exposure of cleaving embryos resulted in more severe developmental defects than exposure of posthatching blastulae. Moreover, mitotic abnormalities were observed following early exposure of embryos to decreased pH. Increased pH, up to 8.6 (approximately 0.5 pH units above normal value), failed to exert any adverse effect on subsequent development. Moreover, an initial pH increase (8.5-8.7) resulted in the final adjustment of culture pH to 8.1-8.2, thus providing optimal conditions for rearing embryos. Two attempts to stabilize culture pH were performed by decreasing gaseous exchanges or by using Tris as a buffering agent. Both approaches appeared to be impractical, thus ruling out any further attempts. The results point out the hazards of acid contamination in restricted bodies of seawater, leading to apparently "moderate" decreases in pH, which can result in severe damage to some marine organisms, both adult and larval forms.

Animals↗

PubMed1978

Methylmercury: some effects on embryogenesis in the Japanese Medaka, Oryzias latipes.

Japanese Medaka (Oryzias latipes) eggs were treated with 40, 60, and 80 parts per billion (ppb) methylmercuric chloride (MMC) for a period of 10 to 25 days in order to observe gross teratogenic and lethal effects caused by methylmercury during embryogenesis. Over the period tested, 40 ppb-treated embryos were less affected than other treated animals. Embryos treated with 60 ppb MMC showed greater effects in that there was a higher death rate and those which survived ranged considerably in size, degree of gross teratogenic defects, and behavior if development was normal enough for hatching to occur. Most did not hatch during the 25-day test period. Embryos treated with 80 ppb MMC were very abnormal in development, being stunted, poorly pigmented, and they showed various eye defects. None hatched in 25 days. Many did not incorporate the heart into the body. Methylmercuric chloride in trace amounts caused various defects and presents a potential hazard to development of embryos of aquatic vertebrates.

Abnormalities, Drug-Induced↗

PubMed1993

Upstream region of the myogenin gene confers transcriptional activation in muscle cell lineages during mouse embryogenesis.

Myogenin, one of the MyoD-related factors containing basic-helix-loop-helix motifs, is transcriptionally activated in skeletal muscle lineages in somites and limb buds during embryogenesis. In an attempt to understand regulatory mechanisms which govern transcriptional activation of the myogenin gene, transgenic mice bearing the lacZ gene driven by the upstream region of the myogenin gene were generated. Stereoscopic visualization of LacZ-positive cells of these transgenic mouse embryos revealed that the upstream region of the myogenin gene conferred its transcriptional activation in cells of the skeletal muscle lineages in somites, limb buds, and visceral arches. Moreover, transient LacZ expression in newly formed somites in addition to its strong activation in myotomal regions of mature somites with a rostro-caudal gradient raised the possibility that myogenin is transcriptionally activated in immature somites before myotome formation.

Animals↗

PubMed1995

Differential display identifies developmentally regulated genes during somatic embryogenesis in eggplant (Solanum melongena L.).

Changes in gene expression patterns during early somatic embryogenesis of eggplant (Solanum melongena L.) were characterized by means of differential display. Eight products of polymerase chain reaction (PCR) derived from newly expressed mRNAs after four days of culture were identified and cloned. One clone, pTM002, was found to contain a 416 bp insert and identical to the proximal arbitrary primer at both ends. Based on the terminal sequences, reverse-transcription PCR (RT-PCR) was carried out to monitor the expression level of pTM002. The results indicated that the message of pTM002 was increased to correlate with the intensities obtained in the differential display. The significance of this method in investigation of differentially expressed genes is also discussed.

Base Sequence↗

PubMed1998

Expression of spliceosome-associated protein 49 is required for early embryogenesis in Caenorhabditis elegans.

Spliceosome-associated protein 49 (SAP 49) is a subunit of the splicing factor SF3b, which is involved in 3' splice site recognition in pre-mRNA splicing. Here we show the Caenorhabditis elegans SAP 49 gene is located in a gene cluster that is transcribed polycistronically with three other upstream genes. Transgenic analysis of expression of the gene cluster under the control of its native promoter revealed that in spite of its predicted essential function for all cells, the C. elegans SAP 49 expression is limited to specific cells in both larval and adult stages. When the endogenous SAP 49 expression was inhibited by microinjection of an antisense RNA, embryos laid by the injected worms ceased to develop at a specific stage of early embryogenesis, indicating that SAP 49 plays an essential role in the development of C. elegans. These results raise the possibility that SAP 49 is a cell-specific, not constitutive, splicing factor.

Animals↗

PubMed2001

Isolation and characterization of Vsx1, a novel mouse CVC paired-like homeobox gene expressed during embryogenesis and in the retina.

Gastrula stage mouse embryo RNA was screened by degenerate RT-PCR to yield a novel paired-like homeobox gene. The open reading frame encoded by the cDNA was most similar to human VSX1. Mouse Vsx1 encodes a protein of 363 amino acid residues that contains a CVC domain that was originally identified as a conserved motif among mouse CHX10, goldfish VSX-1 and C. elegans CEH-10. Linkage analysis showed that mouse Vsx1 mapped to the distal region of chromosome 2. RT-PCR analysis detected mouse Vsx1 transcripts from gastrulation and post-gastrulation stage mouse embryos, suggesting a role for Vsx1 during mouse embryogenesis. Analysis of the eyes of mouse chimeras generated with embryonic stem cells in which a lacZ reporter was targeted to the Vsx1 locus suggested that Vsx1 is expressed in the inner nuclear layer of the retina.

Amino Acid Sequence↗

PubMed2002

The homeodomain-interacting protein kinase 2 gene is expressed late in embryogenesis and preferentially in retina, muscle, and neural tissues.

The HIPK2 protein is a member of a recently identified family of nuclear protein kinases that interact with homeodomain transcription factors and that are well conserved in various organisms. HIPK2 acts as a transcriptional co-repressor of homeoproteins and it is a component of a co-repressor complex containing Groucho and a histone deacetylase. We have recently demonstrated that the HIPK2 protein interacts with HMGA1 proteins in vitro and in vivo and that HIPK2 exerts a potent inhibitory effect on the cell growth of different normal cell lines. Here we demonstrate that the HIPK2 gene is expressed during the latest phases of embryogenesis, and preferentially in retina, in muscle and in neural tissues. Conversely, HIPK2 is ubiquitously expressed in murine and human adult tissues. We have also found that HIPK2 expression is reduced in breast and thyroid carcinomas, suggesting a role of this gene in the process of carcinogenesis.

Animals↗

PubMed1993

Proliferation of chicken myoblasts is regulated by specific isoforms of platelet-derived growth factor: evidence for differences between myoblasts from mid and late stages of embryogenesis.

In the present study we measured the level of PDGF receptor expression by chicken myoblasts and the effect of the three different PDGF isoforms (AA, AB, BB) on DNA synthesis by myoblasts. We examined PDGF receptor expression and function on clonally derived myoblasts in order to eliminate contaminating fibroblasts which are present in myogenic cultures and which bind PDGF. Furthermore, since we have previously shown that fetal myoblasts are replaced with adult myoblasts during late chicken embryogenesis, we compared PDGF receptor expression and function on myoblasts from Embryonic Day 10 (E10, mid development) and from Embryonic Day 19 (E19, late development). We found that all myogenic clones from late embryos (E19) express many receptors for PDGF-BB, far fewer receptors for PDGF-AB, and even fewer, if any, receptors for PDGF-AA. Myoblast clones derived from E10 were more heterogeneous in their PDGF binding pattern ranging from clones similar to E19 clones to clones having very few PDGF binding sites. We also found that both PDGF-AB and PDGF-BB can promote DNA synthesis by clonally derived chicken myoblasts maintained in 2.5% fetal bovine serum whereas PDGF-AA has no detectable effect. Finally, we observed that primary myogenic cultures from E10 and E19 differ strikingly in levels of PDGF binding; E19 cultures bind much more PDGF than do E10 cultures. We conclude that PDGF can enhance the proliferation of chicken myoblasts and that myoblasts responsive to PDGF are more frequent in late than in mid stages of development. We propose that PDGF may be a modulator of myogenesis of adult but not fetal myoblasts.

Animals↗

PubMed1993

Regional specification during embryogenesis in the articulate brachiopod Terebratalia.

The timing of regional specification during embryogenesis in Terebratalia transversa has been examined by isolating animal, vegetal, and lateral halves at developmental stages ranging from prior to fertilization through late gastrula. Animal halves isolated from prior to fertilization through late blastula stages did not gastrulate and did not form endoderm. When these animal halves were isolated from prior to fertilization through early blastula, they formed only ciliated vesicles; however, similar halves from late blastulae frequently formed the ectodermal components of the three larval lobes. Vegetal halves isolated prior to fertilization through late blastula gastrulated, formed endoderm, and differentiated most larval structures; however, comparable isolates from late gastrulae showed a marked decline in the ability to form anterior larval structures. Animal halves isolated at this stage showed a corresponding decline in the ability to form posterior larval structures. These results indicate that during oogenesis, regional differences arise so that the animal half of the oocyte has a much greater ability to form anterior ectodermal structures than do other regions of the oocyte, while only the vegetal region has the capacity to gastrulate. Inductive signals produced by the vegetal region of the embryo act on the animal half to give it the capacity to form ectodermal components of the three larval lobes. This vegetal-inductive influence has played a major role in regional specification by the late blastula stage of development. Pairs of lateral halves isolated from the same oocyte or embryo from prior to fertilization through late blastula frequently gastrulate, form endoderm, and proceed to form all or most of the larval lobes. The late blastula is the first developmental stage where there is a marked decline in the ability of pairs of lateral halves to differentiate larval structures and is the first stage where pairs of halves resembling dorsal and ventral halves appear. This suggests that the axis of bilateral symmetry is also specified by the late blastula stage of development.

Alkaline Phosphatase↗

PubMed1993

Chromatin transitions during early Xenopus embryogenesis: changes in histone H4 acetylation and in linker histone type.

We describe major transitions in the type and modification of chromatin-associated proteins during the early development of Xenopus laevis. Histone H4 is stored in the diacetylated form in the egg and is progressively deacetylated during normal development. If histone deacetylases are inhibited with sodium butyrate, hyperacetylated histone H4 only accumulates after the mid-blastula transition. The type of linker histone in chromatin also changes during embryogenesis, from predominantly the B4 protein at the mid-blastula transition to predominantly histone H1 at the end of gastrulation. These transitions in chromatin composition correlate with major changes in the replicative and transcriptional activity of embryonic nuclei.

Acetylation↗

PubMed1995

Regional specification during embryogenesis in the inarticulate brachiopod Glottidia.

A fate map has been constructed for the lingulid brachiopod Glottidia pyramidata. The animal half of the egg forms part of the apical lobe and the dorsal valve of the larva. The vegetal half of the egg forms mesoderm and endoderm and is the site of gastrulation; it also forms part of the apical lobe and the ventral valve of the larva. The plane of the first cleavage goes through the animal-vegetal axis of the egg along the future plane of bilateral symmetry of the larva. The timing of regional specification in these embryos was examined by isolating animal or vegetal, anterior or posterior, or lateral regions at different times from prior to fertilization through gastrulation. Animal halves isolated at all stages formed an epithelial vesicle and did not gastrulate. When these halves were isolated from unfertilized eggs or early cleavage stage embryos, they usually did not form an apical lobe or valve; however, when the halves were isolated at later developmental stages, these structures differentiated in a high frequency of cases. Vegetal halves were isolated at all stages gastrulated and formed a larva; however, when these halves were isolated at gastrulation they frequently lacked a dorsal valve. When lateral cuts were made along the animal-vegetal axis at all developmental stages, both halves gastrulated. When the cut was made perpendicular to the plane of the first cleavage from the four-cell stage on, one-half formed the anterior end and the other half formed the posterior end of the larva. These results suggest that there are localized determinants in the egg that specify the different regions of the larva, but there is also an inductive signal(s) from the vegetal region of the embryo that is necessary in order for cells that inherit a given determinant to differentiate. Embryogenesis in Glottidia is compared with articulate brachiopods and phoronids.

Animals↗

PubMed1995

Myogenin's functions do not overlap with those of MyoD or Myf-5 during mouse embryogenesis.

The four myogenic basic helix-loop-helix proteins, MyoD, myogenin, Myf-5, and MRF4, can each activate skeletal muscle differentiation when introduced into nonmuscle cells. During embryogenesis, each of these genes is expressed in a unique but overlapping pattern in skeletal muscle precursors and their descendants. Gene knockout experiments have shown that MyoD and Myf-5 play seemingly redundant roles in the generation of myoblasts. However, the role of either of these genes during differentiation in vivo has not been determined. In contrast, a myogenin-null mutation blocks differentiation and results in a dramatic decrease in muscle fiber formation, yet the role of myogenin in the generation or maintenance of myoblast populations is not known. Because myogenin possesses the same myogenic activity as MyoD and Myf-5 in vitro and the expression patterns of these three genes overlap in vivo, we sought to determine if myogenin shares certain functions with either MyoD or Myf-5 in vivo. We therefore generated mice with double homozygous null mutations in the genes encoding MyoD and myogenin or Myf-5 and myogenin. These mice showed embryonic and perinatal phenotypes characteristic of the combined defects observed in mice mutant for each gene alone. As shown by histological analysis and expression of muscle-specific genes, the numbers of undifferentiated myoblasts and residual myofibers were comparable between myogenin-mutant homozygotes and the double-mutant homozygotes. Myoblasts isolated from neonates of the combined mutant genotypes underwent myogenesis in tissue culture, indicating that no more than two of the four myogenic factors are required to support muscle differentiation. These results demonstrate that the functions of myogenin do not overlap with those of MyoD or Myf-5 and support the view that myogenin acts in a genetic pathway downstream of MyoD and Myf-5.

Animals↗

PubMed1995

Cloning of tropomodulin cDNA and localization of gene transcripts during mouse embryogenesis.

Tropomodulin (Tmod) is a tropomyosin-binding protein involved in the structuring of actin filaments. This report describes Tmod expression in distinct patterns during embryonic development in a wider variety of adult and embryonic vertebrate tissues than previously reported. Identical Tmod cDNAs were cloned from mouse brain, skeletal muscle, heart, and hematopoeitic cells. Genomic blotting demonstrates that Tmod is encoded by a single gene, which has a 1077-bp open reading frame that is highly homologous to that of the human erythrocyte. The spatial and temporal expression of the Tmod gene was examined during mouse embryogenesis using in situ hybridization. Tmod mRNA is present by 9.5 days postcoitum (p.c.) in the developing rostral somites, coincident with expression of contractile protein genes in myotomes, suggesting that Tmod may play an important role in sarcomeric thin filament organization in skeletal muscle. While the expression of Tmod mRNA in cardiac muscle is earlier than that in skeletal muscle, its appearance in the heart also coincides with the expression of genes for thin filament proteins and correlates with initial myocardial contractions at 8.0 days p.c. Tmod mRNA is not detected in developing smooth muscle of the gut, but Tmod mRNA is expressed in hematopoeitic cells in yolk sac and developing liver. The sensory ganglia and epithelia of the inner ear express Tmod mRNA as do other sensory neurons such as those in the olfactory epithelium. Expression levels in the brain are much lower prenatally than postnatally. These findings show that Tmod expression in many cell types is developmentally regulated, suggesting that the interaction of actin filaments with this tropomyosin binding protein is an important process in tissue and cell differentiation.

Amino Acid Sequence↗

PubMed1995

Characterization of chicken syndecan-3 as a heparan sulfate proteoglycan and its expression during embryogenesis.

Syndecan-3 is one of four identified members of a family of transmembrane proteoglycans (the syndecans) that possess highly similar cytoplasmic and transmembrane domains and may function as extracellular matrix receptors and/or low affinity receptors for signaling molecules such as FGF. We previously reported the cloning of a partial cDNA for chicken syndecan-3. Here we report the isolation of a syndecan-3 cDNA containing additional 5' sequence which includes a potential methionine start codon and putative signal sequence. In vitro translation of syndecan-3 cDNA in the presence and absence of microsomes suggests that the putative signal sequence is functional, suggesting that the cDNA may encompass the full coding sequence. We also identify syndecan-3 as a heparan sulfate proteoglycan and report its expression pattern during chicken embryogenesis using polyclonal antibodies raised against a recombinant fusion protein. We detect abundant syndecan-3 expression in the developing brain and neural tube, including a striking expression in the floor plate of the neural tube. During limb development, syndecan-3 is expressed in the distal mesenchymal cells of the limb bud which are undergoing outgrowth in response to the apical ectodermal ridge. Syndecan-3 is also transiently expressed during the formation of the precartilage condensations of the skeletal elements of the limb and subsequently in association with the differentiating osteoblasts of the periosteum. Expression is also observed in several areas of tissue interactions including the developing lens, otic vesicle, genital ridge, sclerotome, and feather buds.

Amino Acid Sequence↗