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Functions for PS integrins in tissue adhesion, migration, and shape changes during early embryonic development in Drosophila.

The position-specific (PS) integrins of Drosophila are a family of cell surface heterodimers that function as receptors for extracellular matrix molecules. In this study we used time-lapse videomicroscopy to examine living embryos lacking both maternal and zygotic expression of the genes that encode the alpha PS1, alpha PS2, and beta PS integrin subunits. We demonstrate roles for these molecules as early as gastrulation. Abnormalities in mutant embryos include: separation and twisting of the embryonic germband, abnormal shape and migration of midgut primordia, irregular visceral mesoderm, detachment of amnioserosa cells, rupture of the cuticle along the dorsal midline, lack of midgut constriction, and detachment of somatic muscles. These observations suggest multiple roles for PS integrins in the adhesion of cells and in the formation, organization, and migration of embryonic tissues. We also show that although alpha PS1 beta PS and alpha PS2 beta PS are often expressed in adjacent embryonic tissues, this distribution does not necessarily reflect equivalent requirements. Furthermore, the complete loss of both alpha subunits does not produce all of the phenotypes observed in embryos lacking beta PS. This suggests that alpha PS1 beta PS and alpha PS2 beta PS are not required in all embryonic processes utilizing PS integrins.

Animals↗

Changes in fatty acid compositions of mitochondria during embryonic development.

1. A general trend among biomembranes of hepatocytes in the developing avian embryo is to display increasing percentages of unsaturated fatty acids, especially oleic acid (C18:1). 2. However, once increasing amounts of thyroxine appear in the plasma, mitochondria begin to exhibit increasing percentages of saturated fatty acids, primarily stearic acid (C18:0). 3. Increasing saturation of mitochondrial membrane lipids can be inhibited by exposure of embryonated eggs to 500 R of X-irradiation. 4. Injection of embryonated eggs with estrone increases the proportion of oleic acid (C18:1) in mitochondrial membranes but a balancing increase in palmitic acid (C16:0) enables their lipids to remain more saturated than unsaturated.

Animals↗

Role of Caenorhabditis elegans protein phosphatase type 1, CeGLC-7 beta, in metaphase to anaphase transition during embryonic development.

In Caenorhabditis elegans embryogenesis, phosphorylation events are critical to chromosomal changes. To investigate the dephosphorylation of chromosome behavior, we cloned and characterized the cDNA that encodes C. elegans protein phosphatase type 1 (CeGLC-7 beta), which is composed of 333 amino acids. CeGLC-7 beta possesses a highly conserved amino acid sequence with mammalian and Drosophila protein phosphatase 1. Here, we report on the contribution of CeGLC-7 beta to the dephosphorylation of histone H3 at anaphase. At the embryonic stage, CeGLC-7 beta is associated with the nuclear membrane and chromosomes. The deletion of the Ceglc-7 beta gene and a microinjection of double-stranded RNA produce a disorganized embryogenesis. The Ceglc-7 beta gene mutation causes an abnormal accumulation of phosphorylated histone H3 and delays the mitotic process after anaphase. We propose that CeGLC-7 beta is involved in chromosome dynamics including histone H3 dephosphorylation.

Amino Acid Sequence↗

The embryonic development of the flatworm Macrostomum sp.

Macrostomid flatworms represent a group of basal bilaterians with primitive developmental and morphological characteristics. The species Macrostomum sp., raised under laboratory conditions, has a short generation time of about 2-3 weeks and produces a large number of eggs year round. Using live observation, histology, electron microscopy and immunohistochemistry we have carried out a developmental analysis of Macrostomum sp. Cleavage (stages 1-2) of this species follows a modified spiral pattern and results in a solid embryonic primordium surrounded by an external yolk layer. During stage 3, cells at the anterior and lateral periphery of the embryo evolve into the somatic primordium which gives rise to the body wall and nervous system. Cells in the center form the large yolk-rich gut primordium. During stage 4, the brain primordium and the pharynx primordium appear as symmetric densities anterior-ventrally within the somatic primordium. Organ differentiation commences during stage 5 when the neurons of the brain primordium extend axons that form a central neuropile, and the outer cell layer of the somatic primordium turns into a ciliated epidermal epithelium. Cilia also appear in the lumen of the pharynx primordium, in the protonephridial system and, slightly later, in the lumen of the gut. Ultrastructurally, these differentiating cells show the hallmarks of platyhelminth epithelia, with a pronounced apical assembly of microfilaments (terminal web) inserting at the zonula adherens, and a wide band of septate junctions underneath the zonula. Terminal web and zonula adherens are particularly well observed in the epidermis. During stage 6, the somatic primordium extends around the surface dorsally and ventrally to form a complete body wall. Muscle precursors extend myofilaments that are organized into a highly regular orthogonal network of circular, diagonal and longitudinal fibers. Neurons of the brain primordium differentiate a commissural neuropile that extends a single pair of ventro-lateral nerve trunks (the main longitudinal cords) posteriorly. The primordial pharynx lumen fuses with the ventral epidermis anteriorly and the gut posteriorly, thereby generating a continuous digestive tract. The embryo adopts its final shape during stages 7 and 8, characterized by the morphallactic lengthening of the body into a U-shaped form and the condensation of the nervous system.

Animals↗

The effect of Imuran on implantation and early embryonic development in rats.

Pregnant rats were injected daily with 2, 4, and 20 mg/kg of Imuran from the first day of gestation until sacrificed on Days 6-12. Gravid uteri, as well as maternal organs, were removed and examined histologically. According to the histologic findings, the treatment with the high dose of 20 mg/kg/day Imuran induced injuries to the blastocyst. Starting from Day 8, the trophoblastic layer lost its continuity and maternal blood invaded into the blastocystic cavity resulting in destruction of embryonic tissues. This degeneration progressed until evident resorption on Day 12. Smaller doses, such as 2 or 4 mg/kg of Imuran, did not result in early trophoblastic damage. The effect seemed to be the result of the antimetabolic effect of Imuran acting on the proliferating trophoblast through the maternal blood.

Animals↗

[Time of the action of genes controlling the activity of aldolase in embryonal development of groudling].

The time of action of the genes controlling the decrease of aldolase activity (21-23 hrs of development) and its subsequent increase (23-36 hrs) was determined by means of inactivation of the nuclei by actinomycin or heavy doses of irradiation at succesive developmental stages. There exist two distinct periods of gene activity; the former (15-18 hrs) determines the rapid fall of maternal aldolase activity and the latter (21-27 hrs) its subsequent replacement by embryonic aldolase. This result is confirmed by the data concerning the changes in aldolase heat resistance in the hybrids of the loach and tropical cyprinids. The genes controlling the synthesis of the new aldolase and the morphogenesis which takes place at the same developmental stages are functioning at different times, i.e. the biochemical and morphological differentiations may occur relatively independently.

Animals↗

L-DOPA supply to the neuro retina activates dopaminergic communication at the early stages of embryonic development.

DOPA decarboxylase (DDC; aromatic-l-amino acid decarboxylase; EC 4.1.1.28) is absent in retinas from 6-day-old chicken embryos (E6) but is expressed in retina of E8 embryos, in the presumptive outer plexiform layer. Thereafter, DDC appears in cell bodies of presumptive amacrine cells. The dopamine (DA) content of E9/10 and E15/16 retinas, pre-incubated with l-DOPA for 1 h, increased 250- and 600-fold, respectively, showing that DDC is active since early in development. Intercellular communication, measured by endogenous cyclic AMP accumulation, was observed when retinas from E9/10 to E15/16 were pre-incubated for 1 h with 1 mm l-DOPA, washed and followed by incubation in the presence of 0.5 mm 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. Cyclic AMP accumulation was prevented when pre-incubation with l-DOPA was carried out in the presence of carbidopa. Moreover, the accumulation of cyclic AMP was inhibited by SCH 23390 (2 micro m). The incubation of retinas in medium previously conditioned by retina-pigmented epithelium (RPE) also increased its cyclic AMP content with the characteristics described for l-DOPA. Our results show that dopaminergic communication takes place in the embryonic retina, before tyrosine hydroxylase expression, provided l-DOPA is supplied to the tissue. It also shows that RPE is a potential source of l-DOPA early in development.

Animals↗

Multiple functions of Jab1 are required for early embryonic development and growth potential in mice.

Jab1 interacts with a variety of signaling molecules and regulates their stability in mammalian cells. As the fifth component of the COP9 signalosome (CSN) complex, Jab1 (CSN5) plays a central role in the deneddylation of the cullin subunit of the Skp1-Cullin-F box protein ubiquitin ligase complex. In addition, a CSN-independent function of Jab1 is suggested but is less well characterized. To elucidate the function of Jab1, we targeted the Jab1 locus by homologous recombination in mouse embryonic stem cells. Jab1-null embryos died soon after implantation. Jab1-/- embryonic cells, which lacked other CSN components, expressed higher levels of p27, p53, and cyclin E, resulting in impaired proliferation and accelerated apoptosis. Jab1 heterozygous mice were healthy and fertile but smaller than their wild-type littermates. Jab1+/- mouse embryonic fibroblast cells, in which the amount of Jab1-containing small subcomplex, but not that of CSN, was selectively reduced, proliferated poorly, showed an inefficient down-regulation of p27 during G1, and was delayed in the progression from G0 to S phase by 3 h compared with the wild-type cells. Most interestingly, in Jab1+/- mouse embryonic fibroblasts, the levels of cyclin E and deneddylated Cul1 were unchanged, and p53 was not induced. Thus, Jab1 controls cell cycle progression and cell survival by regulating multiple cell cycle signaling pathways.

Animals↗

Effects of selection for growth and selection diet on eggshell quality and embryonic development in Japanese quail.

Decreased hatchability in Japanese quail following selection for growth and relative improvements in hatchability following selection during goitrogen treatment were investigated. An unselected quail line (Line C) and lines selected for high 4-wk body weight while being fed diets containing either 20% CP and .2% thiouracil (TU) (Line T), or 28% CP and no TU (Lines P and H-CD) were used. Egg weight loss between 0 and 4, and 0 and 14 days of incubation, hatchability, and developmental stage of embryonic mortalities were determined in Experiment 1. Length of the incubation period, and percentages of body water and dry body weight were determined for 10- and 14-day embryos and 18-day-old chicks in Experiment 2. Eggshell quality was determined in Experiment 3. Hatchability was lower in selected than in unselected quail due to increased early and late embryonic mortality. Egg weight loss during incubation was greater in Line P than in Line C eggs. Weight loss, measured across lines, was lower in eggs that hatched than in those in which embryos died early. Percentage body water was higher in Line C than in Lines P and T at 14 and 18 days of incubation; however, percentage body water in Line T was lower than Line P at 14 days. Percentage of dry body weight was greater in Lines P and T than in Line C at 14 and 18 days. Eggshell thickness was greater in Line H-CD when compared with Line C. Increases in eggshell permeability occurring during selection for growth were associated with increased embryonic mortality and decreased hatchability.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Site-specific interactions of neurotrophin-3 and fibroblast growth factor (FGF2) in the embryonic development of the mouse cochlear nucleus.

Neurotrophins and FGF2 contribute to formation of the cochlea, but their roles in cochlear nucleus development are unknown. The effects of these factors may differ in the cochlea and cochlear nucleus, which may influence each other's development. It is important to analyze the effects of these factors on cellular structures at well-defined steps in the normal morphogenetic sequence. The present study used immunohistochemistry to localize factors in situ and to test hypotheses about their roles in an in vitro model. Specific antibody staining revealed that TrkC, the NT3 receptor, is present in neural precursors prior to embryonic day E11 until after birth. NT3 appeared in precursor cells during migration (E13-E15) and disappeared at birth. TrkC and NT3 occurred in the same structures, including growing axons, terminals, and their synaptic targets. Thus, NT3 tracks the migration routes and the morphogenetic sequences within a window defined by TrkC. In vitro, the cochlear nucleus anlage was explanted from E11 embryos. Cultures were divided into groups fed with defined medium, with or without FGF2, BDNF, and NT3 supplements, alone or in combinations, for 7 days. When neuroblasts migrated and differentiated, immunostaining was used for locating NT3 and TrkC in the morphogenetic sequence, bromodeoxyuridine for proliferation, and synaptic vesicle protein for synaptogenesis. By time-lapse imaging and quantitative measures, the results support the hypothesis that FGF2 promotes proliferation and migration. NT3 interacts with FGF2 and BDNF to promote neurite outgrowth, fasciculation, and synapse formation. Factors and receptors localize to the structural sites undergoing critical changes.

Animals↗

Sea urchin small RNA ribonucleoprotein particles: identification, synthesis, and subcellular localization during early embryonic development.

Small RNAs in sea urchins were examined in order to characterize developmental changes in their level, subcellular localization, synthesis, and association with proteins and other RNAs. Small RNAs such as the U snRNAs, 5S and 5.8S rRNAs, and 7S RNAs were identified by their mobility on highly cross-linked acrylamide gels. In addition, 7SL and U1 RNAs were identified by northern blot hybridization to cloned human and sea urchin probes, respectively. The level, subcellular localization, and association with proteins or RNA do not change for most small RNAs from fertilization to blastula, even though this is the time when the stored maternal pool of many small RNAs is being supplemented and replaced by embryonically synthesized RNAs. New embryonic synthesis of small RNAs was first detected at the 8-12 hr blastula stage. Although the predicted subsets of the total small RNA pool can be found in the appropriate subcellular compartments, newly synthesized small RNAs have a predominantly cytoplasmic localization: All of the newly synthesized small RNAs were found to be constituents of small RNPs. The RNPs containing newly synthesized small RNAs had sedimentation rates indistinguishable from their maternal counterparts. Thus, on the basis of sedimentation rate, no gross differences could be detected between maternal and embryonic small RNP pools. These small RNPs include a cytoplasmic RNP containing newly synthesized U1 snRNA and the sea urchin signal recognition particle (SRP) containing the 7SL, RNA. We have also identified a small RNP bearing the 5S rRNA which is present in both eggs and embryos. The presence of multiple, abundant, small RNAs and RNPs that are maintained at constant levels in particular subcellular fractions throughout development suggests that small RNAs may be involved in many more cellular activities than have so far been described.

Animals↗

Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development.

In cell culture assays, Frizzled and Dfrizzled2, two members of the Frizzled family of integral membrane proteins, are able to bind Wingless and transduce the Wingless signal. To address the role of these proteins in the intact organism and to explore the question of specificity of ligand-receptor interactions in vivo, we have conducted a genetic analysis of frizzled and Dfrizzled2 in the embryo. These experiments utilize a small gamma-ray-induced deficiency that uncovers Dfrizzled2. Mutants lacking maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit defects in the embryonic epidermis, CNS, heart and midgut that are indistinguishable from those observed in wingless mutants. Epidermal patterning defects in the frizzled, Dfrizzled2 double-mutant embryos can be rescued by ectopic expression of either gene. In frizzled, Dfrizzled2 mutant embryos, ectopic production of Wingless does not detectably alter the epidermal patterning defect, but ectopic production of an activated form of Armadillo produces a naked cuticle phenotype indistinguishable from that produced by ectopic production of activated Armadillo in wild-type embryos. These experiments indicate that frizzled and Dfrizzled2 function downstream of wingless and upstream of armadillo, consistent with their proposed roles as Wingless receptors. The lack of an effect on epidermal patterning of ectopic Wingless in a frizzled, Dfrizzled2 double mutant argues against the existence of additional Wingless receptors in the embryo or a model in which Frizzled and Dfrizzled2 act simply to present the ligand to its bona fide receptor. These data lead to the conclusion that Frizzled and Dfrizzled2 function as redundant Wingless receptors in multiple embryonic tissues and that this role is accurately reflected in tissue culture experiments. The redundancy of Frizzled and Dfrizzled2 explains why Wingless receptors were not identified in earlier genetic screens for mutants defective in embryonic patterning.

Animals↗

Polymorphism in fowl serum albumin. VI. Changes in in vitro protein synthesizing activity in developing embryonic fowl liver.

Cell-free protein synthesizing systems were prepared from the livers of chick embryos at selected ages and the characteristics of individual fractions were compared. While polysomes showed decreasing size with older embryos, isolated polysomes did not differ significantly in amino acid incorporating activity when assayed with standard cell sap. When assayed with standard polysomes, cell sap activity decreased with increasing developmental age whether incorporation was measured using (3H)lysine, (3H)leucine, or [3H]aminoacyl-tRNA. Free amino acid concentrations in the cell sap showed reproducidble independent variation during development which was taken into consideration in calculating net amino acid incorporation. A larger increase in ribonuclease activity was observed during development; however, nuclease inhibitor activity was absent before day 15 but increased thereafter. Aminoacyl-tRNA sythetase activity did not vary significantly. It is proposed that the observed changes in the rate of cell-free protein synthesis result not only from increasing ribonuclease activity with increasing developmental age but also from changes in the activity of other soluble factors.

Amino Acids↗

The Drosophila EGF receptor homolog (DER) gene is allelic to faint little ball, a locus essential for embryonic development.

Recessive lethal mutations in the genetic locus of the Drosophila EGF receptor homolog (DER) were isolated. Identification of mutations in the gene is based on assays of DER protein autophosphorylation activity. Most DER alleles show little or no in vivo autophosphorylation. The ability to monitor these activities in vivo and in vitro offers a preliminary insight into the functional defects in the different mutant proteins. The identification of the DER locus was also confirmed by partial rescue of the mutant phenotype with a DER P-element construct. Homozygous DER mutants display a complex embryonic phenotype. Most notably, the anterior structures deteriorate, ventral denticle bands are missing, the germ band does not retract, and the central nervous system shows a collapse of commissure and midline pattern. Mutations in DER were shown to be allelic to the previously described locus faint little ball.

Alleles↗

Ontogenesis of physiological responsiveness and guanine nucleotide sensitivity of cardiac muscarinic receptors during chick embryonic development.

Atria isolated from 4-day chick embryos were much less responsive to the negative chronotropic effect of muscarinic agonists than were atria from 5- or 8-day embryos, even though the density of muscarinic acetylcholine receptors (mAChR) was similar at all these ages. The mAChR in hearts from 4-day embryos were also significantly less susceptible to regulation of receptor number by in vivo agonist treatment and required a 2-5-fold greater dose of the muscarinic agonist carbachol to achieve a decrease in receptor number equivalent to that observed in 5- or 8-day embryonic hearts. When 4-day atrial membranes were assayed in physiological buffers, agonist binding to the mAChR was not regulated by GTP unless a sulfhydryl reducing agent was present. Receptors from 5- and 8-day embryos did not require addition of a sulfhydryl reducing agent in order to see guanine nucleotide effects on agonist binding. Even in the presence of a sulfhydryl reducing agent, carbachol binding to the mAChR in 4-day membranes was much less sensitive to guanyl-5'-yl imidodiphosphate (GppNHp) than binding to mAChR in 5- or 8-day membranes. In addition, forskolin-activated adenylate cyclase activity was much less sensitive to inhibition by GppNHp in membranes from 4-day atria than from 5- and 8-day atria. The GTP-binding component (NI) which couples the mAChR to inhibition of adenylate cyclase activity was examined by covalent modification with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Cyclase Toxin↗

Embryonic development in the primitive bilaterian Neochildia fusca: normal morphogenesis and isolation of POU genes Brn-1 and Brn-3.

Neochildia fusca is a member of the taxon Acoela, a group of flatworms that, according to some recent molecular phylogenetic analyses, are distinct from other flatworms and constitute a basal branch with a sister taxon relationship to the rest of the Bilateria. In this paper, we analyze early neural development in this species and report the sequence and expression of two Pit-Oct-Unc (POU) genes, NeocBrn-1 and NeocBrn-3. Homologs of these highly conserved genes play a role in neural fate determination in vertebrates, Drosophila and Caenorhabditis elegans. Acoels, including Neochildia, have a unique invariant pattern of early cleavage called duet spiral cleavage. In subsequent cell divisions descendants of the first three micromere duets form an outer layer of epidermal and neural progenitors surrounding the meso/endoderm progenitors, which are themselves descended from the macromere duet 4A, B and the micromere duet 4a, b. Organ formation begins at mid-embryonic stages with the epidermal primordium adopting a ciliated epithelial shape. Sub-epidermally, a bilaterally symmetric brain primordium can be seen at the anterior pole. Laterally and posteriorly, myoblasts form a thin layer underneath the epidermis. In late embryos and juveniles of Neochildia, the brain is formed by a 3-4 cell-diameter-thick layer of neurons forming a cortex surrounding a neuropile that is relatively free of cell bodies. A highly regular "orthogonal" array of muscle fibers penetrates the brain. We have isolated and partially sequenced homologs of the vertebrate Brn-1 and Brn-3 genes, which we call NeocBrn-1 and NeocBrn-3, respectively. These sequences contain and span portions of the POU-specific domain and a homeodomain, and are sequence similar to their respective homologs in vertebrates and Drosophila. RT-PCR reveals that NeocBrn-1 and NeocBrn-3 are expressed from mid-embryonic to adult stages. Whole-mount in situ hybridization shows expression of both genes in distinct subsets of nerve cells in juvenile and adult worms. NeocBrn-1 also appears in a subset of intra-epidermal gland cells. These observations are an initial step towards reconstructing the neural development of a key group of bilaterians, the Acoela. These flatworms, by virtue of their distinct morphology, development and phylogenetically basal placement, are likely to provide key insights into the interpretation of the evolution of metazoan neural architecture.

Amino Acid Sequence↗

Embryonic development of the myenteric nerve plexus in the rat small intestine as revealed by light and scanning electron microscopy.

This study was performed to clarify developmental changes in the structure of the myenteric nerve plexus in the rat small intestine. The small intestines of fetal and neonatal rats were examined by light microscopic immunohistochemistry for protein gene product 9.5 (PGP-9.5) and alpha-smooth muscle actin (alpha-SMA). Direct observation of the three-dimensional structure of the neuronal and muscular elements was accomplished by scanning electron microscopy of tissues after KOH treatment. At the embryonic day 14 (E14), PGP-9.5 immunopositive (neuronal) cells first appeared in the mesenchymal tissue outside an alpha-SMA positive (muscle) cell layer. These cells increased in number to form a thin and continuous sheet, which was sandwiched between the inner and outer muscular layers at E17. This neuronal layer consisted of immature ganglion cells associated with supportive (enteroglial) cells, and was gradually segregated into groups by incomplete separation through slit-like interruptions at E18. With the widening of these slits at E19, the neuronal sheet was observed as a ladder composed of transversely elongated ganglia and their connecting nerve strands, resulting in the typical structure of the myenteric nerve plexus. Fibroblastic cells of a peculiar shape appeared on the outer surface of the myenteric plexus at E19. These cells (presumably interstitial cells) interconnected their long branching projections to form a cellular network on the myenteric plexus until birth.

Animals↗

Post-transcriptional regulation of Xwnt-8 expression is required for normal myogenesis during vertebrate embryonic development.

The Xenopus Wnt-8 gene is transiently expressed in ventral and lateral mesoderm during gastrulation and plays a critical role in patterning these tissues. In the current study, we show that the spatial and temporal pattern of expression of endogenous Xwnt-8 is regulated, in part, at a post-transcriptional level. We have identified a novel sequence element in the 3' untranslated region of the Xwnt-8 RNA that controls the polyadenylation status of reporter and endogenous Xwnt-8 RNAs, directs rapid RNA degradation beginning precisely at the early gastrula stage, and represses translation of transcripts throughout development. Expression of endogenous Xwnt-8 is normally downregulated within lateral (presomitic) mesoderm following gastrulation. We demonstrate that rapid degradation of Xwnt-8 transcripts, mediated by these regulatory elements in the 3' untranslated region, is essential to this process and that downregulation is required to prevent overcommitment of somitic cells to a myogenic fate. These studies demonstrate a role for post-transcriptional regulation of zygotic gene expression in vertebrate embryonic patterning.

3' Untranslated Regions↗