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Expression of the c-ret proto-oncogene during mouse embryogenesis.

The c-ret proto-oncogene encodes a receptor tyrosine kinase whose normal function has yet to be determined. To begin to investigate the potential role of this gene in vertebrate development, we have isolated cDNA clones representing the murine c-ret gene, and have analyzed the pattern of expression during mouse embryogenesis, using northern blotting, in situ hybridization to histological sections and whole-mount hybridization histochemistry. c-ret transcripts were detected beginning at day 8.5 of embryogenesis, and were observed in a number of cell lineages in the developing peripheral and central nervous systems, as well as in the excretory system. In the cranial region at day 8.5-9.5, c-ret mRNA was restricted to a population of neural crest cells migrating from rhombomere 4 and forming the anlage of the facioacoustic ganglion, as well as to a closely associated domain of surface ectoderm and pharyngeal endoderm. At later stages (10.5-14.5 days), c-ret mRNA was observed in all cranial ganglia. In the peripheral nervous system of the trunk, c-ret was expressed in the autonomic ganglia and in subsets of cells in the dorsal root ganglia. In the enteric nervous system, c-ret was expressed in the presumptive enteric neuroblasts of the vagal crest (day 9.0-11.5), and in the myenteric ganglia of the gut (day 13.5-14.5). c-ret mRNA was observed in several regions of the central nervous system, including the undifferentiated neuroepithelial cells of the ventral neural tube (8.5 days), the motor neurons in the spinal cord and the hindbrain (10.5-14.5 days), the embryonic neuroretina (day 13.5) and the layers of the postnatal retina containing ganglion, amacrine and horizontal cells. Outside the nervous system, c-ret was expressed in the nephric (Wolffian) duct at day 8.5-10.5, the ureteric bud epithelium (but not the surrounding metanephric mesenchyme) at day 11.0-11.5, and the growing tips of the renal collecting ducts (but not the previously formed, subcortical portions of the collecting ducts, or the mesenchyme-derived renal vesicles) at day 13.5-17.5. Our results suggest that the c-ret gene may encode the receptor for a factor involved in the proliferation, migration, differentiation or survival of a variety of neuronal cell lineages, as well as in inductive interactions during organogenesis of the kidney.

Animals↗

Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis.

Members of the MEF2 family of transcription factors bind a conserved A/T-rich sequence in the control regions of many skeletal and cardiac muscle genes. To begin to assess the roles of the different Mef2 genes in the control of muscle gene expression in vivo, we analyzed by in situ hybridization the expression patterns of the Mef2a, Mef2c and Mef2d genes during mouse embryogenesis. We first detected MEF2C expression at day 7.5 postcoitum (p.c.) in cells of the cardiac mesoderm that give rise to the primitive heart tube, making MEF2C one of the earliest markers for the cardiac muscle lineage yet described. By day 8.5, MEF2A, MEF2C and MEF2D mRNAs are all detected in the myocardium. By day 9.0, MEF2C is expressed in rostral myotomes, where its expression lags by about a day behind that of myf5 and several hours behind that of myogenin. MEF2A and MEF2D are expressed at a lower level than MEF2C in the myotome at day 9.5 and are detected in more embryonic tissues than MEF2C. Expression of each of the MEF2 transcripts is observed in muscle-forming regions within the limbs at day 11.5 p.c. and within muscle fibers throughout the embryo at later developmental stages. The expression of MEF2C in the somites and fetal muscle is distinct from that of MEF2A, MEF2D and the myogenic bHLH regulatory genes, suggesting that it may represent a distinct myogenic cell type. Neural crest cells also express high levels of MEF2 mRNAs between days 8.5 and 10.5 of gestation. After day 12.5 p.c., MEF2 transcripts are detected at high levels in specific regions of the brain and ultimately in a wide range of tissues. The distinct patterns of expression of the different Mef2 genes during mouse embryogenesis suggest that these genes respond to unique developmental cues and support the notion that their products play roles in the regulation of muscle-specific transcription during establishment of the cardiac and skeletal muscle lineages.

Animals↗

Apoptosis of the midline glia during Drosophila embryogenesis: a correlation with axon contact.

We have examined cell death within lineages in the midline of Drosophila embryos. Approximately 50% of cells within the anterior, middle and posterior midline glial (MGA, MGM and MGP) lineages died by apoptosis after separation of the commissural axon tracts. Glial apoptosis is blocked in embryos deficient for reaper, where greater than wild-type numbers of midline glia (MG) are present after stage 12. Quantitative studies revealed that MG death followed a consistent temporal pattern during embryogenesis. Apoptotic MG were expelled from the central nervous system and were subsequently engulfed by phagocytic haemocytes. MGA and MGM survival was apparently dependent upon proper axonal contact. In embryos mutant for the commissureless gene, a decrease in axon-glia contact correlated with a decrease in MGA and MGM survival and accelerated the time course of MG death. In embryos mutant for the slit gene, MGA and MGM maintained contact with longitudinally and contralaterally projecting axons and MG survival was comparable to that in wild-type embryos. The initial number of MG within individual ventral nerve cord segments was increased by ectopic expression of the rhomboid gene, without changing axon number. Extra MGA and MGM were eliminated from the ventral nerve cord by apoptosis to restore wild-type numbers of midline glia. Ectopic rhomboid expression also shifted MGA and MGM cell death to an earlier stage of embryogenesis. One possible explanation is that axon-glia contact or communication promotes survival of the MG and that MG death may result from a competition for available axon contact.

Animals↗

Reassessing embryogenesis in the Ctenophora: the inductive role of e1 micromeres in organizing ctene row formation in the 'mosaic' embryo, Mnemiopsis leidyi.

Ctenophores are a phylum of diploblastic marine animals displaying biradial symmetry organized along an oral-aboral axis. One of the apomorphic sets of adult structures in ctenophores are the eight external comb rows, which run along the oral-aboral axis. Comb rows consist of serial arrays of individual comb plates of cilia, which beat in a coordinated fashion for locomotory behavior. Classical cell lineage experiments using chalk particles indicated that comb rows are derived exclusively from the four e1 micromeres at the 16-cell stage. This conclusion was also supported by the fact that no ctene rows (or their underlying endodermal canals) form when all four e1 micromeres were deleted. We have used intracellular diI cell lineage tracing to determine that, in addition to e1 micromeres, the four m1 micromeres also make significant contributions to the ctene rows. Thus, e1 micromere derivatives not only generate comb plates but are required for ctene row formation by m1 derivatives. These results demonstrate that inductive interactions are an important component of early development in ctenophores and indicate that e1 micromeres influence the development of adjacent cell lineages (both m1 and endodermal lineages) during ctenophore embryogenesis. In addition, intracellular labeling has revealed that there are subtle variations in the composition of clones derived from identified embryonic blastomeres. Together these findings reveal a picture of ctenophore embryogenesis, which is in marked contrast to the former rigid 'mosaic' reputation of ctenophore development, and invite speculation as to the role of the cleavage program in embryonic patterning in the lower Metazoa.

Animals↗

Homologous association of the Bithorax-Complex during embryogenesis: consequences for transvection in Drosophila melanogaster.

Transvection is the phenomenon by which the expression of a gene can be controlled by its homologous counterpart in trans, presumably due to pairing of alleles in diploid interphase cells. Transvection or trans-sensing phenomena have been reported for several loci in Drosophila, the most thoroughly studied of which is the Bithorax-Complex (BX-C). It is not known how early trans-sensing occurs nor the extent or duration of the underlying physical interactions. We have investigated the physical proximity of homologous genes of the BX-C during Drosophila melanogaster embryogenesis by applying fluorescent in situ hybridization techniques together with high-resolution confocal light microscopy and digital image processing. The association of homologous alleles of the BX-C starts in nuclear division cycle 13, reaches a plateau of 70% in postgastrulating embryos, and is not perturbed by the transcriptional state of the genes throughout embryogenesis. Pairing frequencies never reach 100%, indicating that the homologous associations are in equilibrium with a dissociated state. We determined the effects of translocations and a zeste protein null mutation, both of which strongly diminish transvection phenotypes, on the extent of diploid homologue pairing. Although translocating one allele of the BX-C from the right arm of chromosome 3 to the left arm of chromosome 3 or to the X chromosome abolished trans-regulation of the Ultrabithorax gene, pairing of homologous alleles surprisingly was reduced only to 20-30%. A zeste protein null mutation neither delayed the onset of pairing nor led to unpairing of the homologous alleles. These data are discussed in the light of different models for trans-regulation. We examined the onset of pairing of the chromosome 4 as well as of loci near the centromere of chromosome 3 and near the telomere of 3R in order to test models for the mechanism of homologue pairing.

Alleles↗

Roles of the Homothorax/Meis/Prep homolog UNC-62 and the Exd/Pbx homologs CEH-20 and CEH-40 in C. elegans embryogenesis.

Co-factor homeodomain proteins such as Drosophila Homothorax (Hth) and Extradenticle (Exd) and their respective vertebrate homologs, the Meis/Prep and Pbx proteins, can increase the DNA-binding specificity of Hox protein transcription factors and appear to be required for many of their developmental functions. We show that the unc-62 gene encodes the C. elegans ortholog of Hth, and that maternal-effect unc-62 mutations can cause severe posterior disorganization during embryogenesis (Nob phenotype), superficially similar to that seen in embryos lacking function of either the two posterior-group Hox genes nob-1 and php-3 or the caudal homolog pal-1. Other zygotically acting unc-62 alleles cause earlier embryonic arrest or incompletely penetrant larval lethality with variable morphogenetic defects among the survivors, suggesting that unc-62 functions are required at several stages of development. The differential accumulation of four unc-62 transcripts is consistent with multiple functions. The C. elegans exd homologs ceh-20 and ceh-40 interact genetically with unc-62 and may have overlapping roles in embryogenesis: neither CEH-20 nor CEH-40 appears to be required when the other is present, but loss of both functions causes incompletely penetrant embryonic lethality in the presence of unc-62(+) and complete embryonic lethality in the presence of an unc-62 hypomorphic allele.

Alternative Splicing↗

Cathepsin L protease (CPL-1) is essential for yolk processing during embryogenesis in Caenorhabditis elegans.

Cysteine proteases are involved in the degradation of intracellular and extracellular proteins, although their precise roles in vivo are not well understood. Here we characterise a genetic mutant of the Caenorhabditis elegans cathepsin L protease gene cpl-1. CPL-1 is provided maternally and is essential for C. elegans embryogenesis. Immunofluorescence and electron microscopy data show that yolk endocytosis and initial yolk platelet formation occur normally in cpl-1 mutant oocytes and embryos. However, at around the 8-12 cell stage of embryogenesis, yolk platelets begin to aggregate and these enlarged yolk platelets fill the cytoplasm of cpl-1 mutant embryos. Coincident with this aggregation is loss of fluorescence from a yolk green fluorescent protein (YP170::GFP). This suggests that loss of CPL-1 activity leads to aberrant processing and/or conformational changes in yolk proteins, resulting in abnormal platelet fusion. This study has relevance to the abnormal fusion and aggregation of lysosomes in cathepsin L-deficient mice and to other lysosomal disorders.

Animals↗

Cytological characterisation of the mutant phenotypes produced during early embryogenesis by null and loss-of-function alleles of the gammaTub37C gene in Drosophila.

We have studied the mutant phenotypes brought about during early embryogenesis by mutation in the gammaTub37C gene, one of the two isoforms of gamma-tubulin that have been identified in Drosophila. We have focused our attention on fs(2)TW1(1) and fs(2)TW1(RU34), a null and a hypomorph allele of this gene, whose sequences we report in this work. We have found that the abnormal meiotic figures observed in mutant stage 14 oocytes are not observed in laid oocytes or fertilised embryos, suggesting that these abnormal meiotic figures are not terminally arrested. We have also concluded that both null and hypomorph alleles lead to a total arrest of nuclear proliferation during early embryogenesis. This is in contrast to their effect on female meiosis-I where hypomorph alleles display a much weaker phenotype. Finally, we have observed that null and hypomorph alleles lead to some distinct phenotypes. Unfertilised laid oocytes and fertilised embryos deficient for gammaTub37C do not contain polar bodies and have a few bipolar microtubule arrays. In contrast, oocytes and embryos from weaker alleles do not have these microtubule arrays, but do contain polar bodies, or polar-body-like structures. These results indicate that gammaTub37C is essential for nuclear proliferation in the early Drosophila embryo.

Alleles↗

Calcium-regulated fusion of yolk granules is important for yolk degradation during early embryogenesis of Rhodnius prolixus Stahl.

This study examined the process of membrane fusion of yolk granules (YGs) during early embryogenesis of Rhodnius prolixus. We show that eggs collected at days 0 and 3 after oviposition contain different populations of YGs, for example day-3 eggs are enriched in large YGs (LYGs). Day-3 eggs also contain the highest free [Ca(2+)] during early embryogenesis of this insect. In vitro incubations of day-0 YGs with [Ca(2+)] similar to those found in day-3 eggs resulted in the formation of LYGs, as observed in vivo. Fractionation of LYGs and small YGs (SYGs) and their subsequent incubation with the fluorescent membrane marker PKH67 showed a calcium-dependent transference of fluorescence from SYGs to LYGs, possibly as the result of membrane fusion. Acid phosphatase and H(+)-PPase activities were remarkably increased in day-3 LYGs and in calcium-treated day-0 LYGs. Both fractions were found to contain vitellins as major components, and incubation of YGs with calcium induced yolk proteolysis in vitro. Altogether, our results suggest that calcium-induced membrane fusion events take part in yolk degradation, leading to the assembly of the yolk mobilization machinery.

Animals↗

Experimental study of the effect of a series of phosphoroorganic pesticides (Dipterex and Imidan) on embryogenesis.

Experiments conducted on pregnant Wistar rats show that chlorophos (Dipterex) has embroyotoxic and teratogenic effects after oral introduction in a 80 mg/kg dose during a critical period of embryogenesis. Embryotoxic and teratogenic effects are absent during the introduction of 8 mg/kg of the pesticide. The oral introduction of phthalophos (Imidan) in a 30 mg/kg dose once on day 9 of pregnancy and the introduction of a 1.5 mg/kg dose daily throughout the course of pregnancy caused increased postimplantation mortality of embryos. A dose of 30 mg/kg of phthalophos on day 9 or day 13 of pregnancy causes developmental abnormalities, including hyponathia and hydrocephaly. A 0.06 mg/kg phthalophos dose does not affect the course of embryogenesis in white rats. Thus the organophosphate pesticides Dipterex and Imidan exhibit embryotoxic and teratogenic effects at doses which significantly exceed the acutal amounts of the pesticide that can enter the human organism.

Animals↗

Hsp70 expression and function during embryogenesis.

This review focuses on the expression and function of 70-kDa heat shock proteins (Hsp70s) during mammalian embryogenesis, though many features of embryogenesis and the developmental expression of Hsp70s are conserved between mammals and other vertebrates. A variety of Hsp70s are expressed from the point of zygotic gene activation in cleavage-stage embryos, through blastulation, implantation, gastrulation, neurulation, organogenesis, and on throughout fetal maturation. The regulation and patterns of hsp70 gene expression and the known and putative Hsp70 protein functions vary from constitutive and metabolic housekeeping to stress-inducible and embryo-protective roles. Understanding the genetic regulation and molecular function of Hsp70s has been pursued by developmental biologists interested in the control of gene expression in early embryos as well as reproductive toxicologists and teratologists interested in how Hsp70s protect embryos from the adverse effects of environmental exposures. These efforts have also been joined by those interested in the chaperone functions of Hsp70s, and this confluence of effort has yielded many advances in our understanding of Hsp70s during critical phases of embryonic development and cellular differentiation.

Animals↗

Direct somatic embryogenesis in Coffea canephora.

Somatic embryogenesis (SE) provides a useful model to study embryo development in plants. In contrast to zygotic embryogenesis, SE can easily be observed, the culture conditions can be controlled, and large quantities of embryos can be easily obtained. In Coffea spp several model systems have been reported for in vitro SE induction. SE for coffee was first reported in Coffea canephora. Several systems have been developed since then, including SE from callus cultures derived from leaf explants; a two-phase experimental protocol for SE from leaves of Coffea arabica; and from leaf explants of Arabusta or C. arabica using a medium with cytokinins. Here we report a protocol using young leaves from in vitro seedling pre-conditioned with growth regulators. This is a simplified method to obtain a faster and more efficient protocol to produce direct somatic embryos in C. canephora.

Coffea↗

Chloroplast genetic engineering via organogenesis or somatic embryogenesis.

Chloroplast genetic engineering offers a number of unique advantages, including high-level transgene expression, multigene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer desired agronomic traits or express high levels of vaccine antigens and biopharmaceuticals. Despite such significant progress, this technology has not been extended to other important plant species. For example, Arabidopsis may be an ideal model system for chloroplast functional genomics. The employment of chloroplast transformation technology in Arabidopsis has been hampered by the lack of an efficient and reproducible protocol that provides fertile chloroplast transgenic plants. Transformation of the Arabidopsis chloroplast genome was achieved via organogenesis but the efficiency was at least a 100-fold lower than in tobacco and had the drawback of polyploidy in the leaf tissue that resulted in sterile transgenic plants. This problem can be overcome by adapting procedures that are now available to regenerate plants from both diploid and tetraploid explants via callus. In addition, it is feasible to regenerate Arabidopsis via somatic embryogenesis. Recent breakthroughs in highly efficient plastid transformation of recalcitrant crops such as cotton and soybean have opened the possibility of engineering Arabidopsis plastid genome via somatic embryogenesis. Therefore, protocols of recent improvements in tissue culture, DNA delivery, and the novel vector designs are provided here in order to achieve highly efficient plastid transformation in Arabidopsis.

Arabidopsis↗

Changes of serum thyroid hormone levels induce malformations on early embryogenesis in rats.

The incidence of malformation is increased in infants of hyperthyroid or hypothyroid woman. Although many papers reported that the fetus is insulted from maternal thyroid hormone, the placenta (maternal-fetal barrier) is not yet fully developed before 11.5 days of gestation in rat embryos, suggesting the effect of thyroid hormone on early rat embryogenesis. This study was, therefore, undertaken to investigate whether excess or lack of thyroid hormones would affect early embryogenesis in rat embryo culture. Malformations including open neuropore and microencephaly were observed in 10 of 30 embryos incubated in hyperthyroid serum, and in 12 of 42 cultured in T3-enriched normal serum. Similar malformations were observed on 14 of 42 embryos cultured in hypothyroid serum and in 10 of 30 cultured in hypothyroid serum supplemented with T3. The frequencies of these malformations were significantly higher than in the control embryos (0 in 72 embryos) cultured with normal rat serum. These results suggest that the maternal thyroid status might play an important role for the complication of fetal malformations during early gestational period.

Animals↗

Anopheles albitarsis embryogenesis: morphological identification of major events.

Anopheles albitarsis embryogenesis was analyzed through confocal microscopy of clarified eggs. Using Drosophila melanogaster as reference system, the major morphogenetic events (blastoderm, gastrulation, germ band extension, germ band retraction, dorsal closure) were identified. The kinetics of early events is proportionally similar in both systems, but late movements (from germ band retraction on) progress slower in An. albitarsis. Major differences in An. albitarsis related to D. melanogaster were: (1) pole cells do not protrude from the blastoderm; (2) the mosquito embryo undergoes a 180 degrees rotation movement, along its longitudinal axis; (3) the head remains individualized throughout embryogenesis; (4) extraembryonary membranes surround the whole embryo. A novel kind of malaria control is under development and is based on the use of genetically modified mosquitoes. Phenotypic analysis of the embryonic development of mutants will be imposed as part of the evaluation of effectiveness and risk of employment of this strategy in the field. In order to accomplish this, knowledge of the wild type embryo is a prerequisite. Morphological studies will also serve as basis for subsequent development biology approaches.

Animals↗

Embryogenesis in hydra.

Embryogenesis in hydra includes a variable period of dormancy; and this period, as well as subsequent stages through hatching, takes place within a thick cuticle that hinders observation. Thus, although the early stages of development have been well-characterized qualitatively, the middle and later stages are only poorly understood. Here, we provide a detailed description of the stages of embryogenesis, including the time required to traverse each of the stages, and the changes that occur in the type and number of cells throughout the stages. The events of cleavage and gastrulation occur within the first 48 h. Cleavage is holoblastic and unipolar and leads to a single-layered coeloblastula. Gastrulation occurs by ingression and is followed by the deposition of the thick cuticle. Thereafter, during the variable period of dormancy ranging from 2-24 weeks, little occurs; the important events are the conversion of the outer layer into an ectoderm and the appearance of the interstitial cell lineage. During the last 2 days before hatching, the endoderm and gastric cavity form, while stem cells of the interstitial cell lineage proliferate and differentiate into neurons, nematocytes, and secretory cells. Finally, the cuticle cracks, and the hatchling enlarges and emerges from the cuticle as a functional animal. The formation of the gastric cavity and the hatching of the embryo are both explicable in terms of the osmotic behavior of the animal and the hydrostatic forces generated by this behavior. Characteristics of development that are common to hydra and triploblastic phyla are presented.

Animals↗

Maternal phenylketonuria-chronology of the detrimental effects on embryogenesis and fetal development: pathological report, survey, clinical application.

Maternal phenylketonuria (PKU) is likely to have detrimental effects on embryogenesis and fetal development. Manifestations in the offspring include spontaneous abortion, various congenital malformations, intrauterine growth retardation, and microcephaly. The time at which the metabolic abnormalities induce pathologic embryogenesis can be documented by knowing the time of the development of specifically damaged organ systems. This review reveals that, while the most recognized congenital malformations occur in the heart, the most common abnormality is growth inhibition occurring throughout pregnancy. The organ system most commonly affected by this growth inhibition is the brain, resulting in a high incidence of micrencephaly. It appears that maternal phenylketonuria interferes with appropriate fetal growth and that this effect occurs during the entire course of pregnancy and has no tissue specificity. This information can be both informative to pathologists and useful to clinicians.

Embryonic and Fetal Development↗

Expression patterns of esophageal cancer deregulated genes in C57BL/6J mouse embryogenesis.

AIM: To investigate the expression patterns of esophageal squamous cell cancer deregulated genes in mid to late stages of C57BL/6J mouse embryogenesis, and the correlation between these genes in embryonic development and tumorigenesis of esophageal squamous cell cancer. METHODS: Reverse northern screening was performed to examine the expression patterns of esophageal cancer deregulated genes in C57BL/6J mouse embryogenesis. To confirm the gene expression patterns, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out for 3 of the randomly picked differentially expressed genes. RESULTS: Within these esophageal cancer deregulated genes, 4 patterns of expression were observed at 3 stages embryonic d 11.5 (E11.5), embryonic d 13.5 (E13.5) and postnatal d1 (P1). (1) Up-regulation during the E11.5 period, down- regulation during the E13.5 and P1 period (up-down-down), the 10 up-regulated genes during the E11.5 period could be classified into 6 known genes and 4 unknown genes. The known genes included differentiation related genes (S100A8), immunity related gene (IGL), translation and transcription regulation genes (RPL15, EEF1A1), cytoskeletal protein (TUBA1), cysteine protease inhibitor (cystatin B). (2) Up-regulation during the E13.5 and P1 period (down-up-up), such as the SPRR2A which was down-regulated at E11.5. (3) Down-regulation during the E11.5 and E13.5 period (down-down-up), such as RHCG and keratin 4. (4) Fluctuating expression, down initially, up at E13.5, and then down again (down-up-down). EMP1 belonged to such a gene, which was highly expressed at E13.5. CONCLUSION: The results will be helpful for understanding the function of esophageal squamous cell carcinoma (ESCC) deregulated genes in embryonic development and tumorigenesis. S100A8 and S100A9 may play different roles in early embryonic development. IGL may be an oncofetal protein, and EMP1 relates with neurogenesis at E13.5. The genes identified pertinent to embryonic development may serve as candidate susceptibility genes for inherited esophageal cancer disorders as well as for various heritable disorders of embryonic development.

Animals↗