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A cell marker system and mosaic patterns during early embryonic development in Drosophila melanogaster.

An embryonic cell marker system has been developed in Drosophila melanogaster that has enabled us to identify the genotype of cells as early as the cellular blastoderm stage of development. This system allows unambiguous detection of embryos homozygous for most X-linked lethal mutations at stages prior to when their first defects become obvious. By examining gynandromorphs at this stage, we have observed that the number of nuclei per unit area in male regions is about half that in female regions. An examination of early cleavage stage embryos whose DNA has been stained with Hoechst 33258 and whose actin has been stained with phalloidin suggests that this difference is due to a cell cycle delay in cells losing the ring-X. These experiments also demonstrate the existence of a mechanism which controls the timing of nuclear divisions in cycle 10-14 embryos.

Animals↗

Influence of the apolipoprotein E epsilon4 allele on human embryonic development.

Human apolipoprotein E (apoE) exists in three major isoforms encoded by distinct alleles (APOE epsilon2, epsilon3 and epsilon4) and has important functions in nerve development and repair. Inheritance of the 4 allele is a major risk factor for the development of Alzheimer's disease. To investigate the role of APOE polymorphisms in embryonic development, we analyzed the APOE genotypes of 81 spontaneously aborted embryos and 110 adult controls using a solid-phase minisequencing technique. The epsilon4 allele was significantly less frequent in the spontaneous abortion group than in the control group (P=0.009), while the frequency of epsilon3 was significantly increased (P=0.005), suggesting that epsilon4 may have protective effects during embryogenesis. These protective effects might counterbalance the deleterious age-related effects of the epsilon4 allele in natural selection.

Apolipoprotein E2↗

Molecular analysis of a cellular decision during embryonic development of Drosophila melanogaster: epidermogenesis or neurogenesis.

In Drosophila melanogaster, the neuroblasts (neural progenitor cells) develop from a special region of the ectoderm, called the neuroectoderm. During early embryonic development, the neuroblasts separate from the remaining cells of the neuroectoderm, which develop as epidermoblasts (epidermal progenitor cells). The separation of these two cell types is the result of cellular interactions. The available data indicate that a signal chain formed by the products of several identified genes regulates the cell's decision to enter either neurogenesis or epidermogenesis. Various kinds of data, in particular from cell transplantation studies and from genetic and molecular analyses, suggest that the proteins encoded by the genes Notch and Delta interact at the membrane of the neuroectodermal cells to provide a regulatory signal. This signal is thought to lead, on the one hand, to epidermal development through the action of the genes of the Enhancer of split complex, a gene complex that encodes several functions related to the transduction and further processing of the signal, including the genetic regulation in the receiving cell; on the other hand, the signal is thought to lead to neural development through the participation of the genes of the achaete-scute complex and daughterless, which are members of a family of DNA-binding regulatory proteins and of the gene vnd whose molecular nature is still unknown.

Animals↗

The Caenorhabditis elegans orphan nuclear hormone receptor gene nhr-2 functions in early embryonic development.

We have identified a Caenorhabditis elegans gene, nhr-2, that is a member of the nuclear hormone receptor superfamily of transcription factors and defines a new subclass of the superfamily. nhr-2 messenger RNA is expressed in the maternal germline and during the first half of embryogenesis. Zygotic expression of nhr-2 begins by the 16-cell stage, making it one of the earliest genes known to be transcribed in the embryo. Immunolocalization detects NHR-2 protein in embryonic nuclei as early as the 2-cell stage. The protein is present in every nucleus until the 16- to 20-cell stage. Subsequently, expression continues in many, but not all, cell lineages, becoming progressively restricted to the anterior and dorsal regions of the embryo and disappearing during the initial stages of morphogenesis. Disruption of nhr-2 function with antisense RNA results in embryonic and early larval arrest, indicating that the gene has an essential function in embryonic development. nhr-2 does not correspond to known mutations mapped to the same genetic interval, and will provide an entry point for further study of a heretofore uncharacterized zygotic gene regulatory pathway.

Amino Acid Sequence↗

Protein distribution of Kcnq1, Kcnh2, and Kcne3 potassium channel subunits during mouse embryonic development.

Voltage-dependent potassium channels consist of a pore-forming alpha-subunit, which is modulated by additional beta-ancillary or regulatory subunits. Kcnq1 and Kcnh2 alpha-channel subunits play pivotal roles in the developing and adult heart. However, Kcnq1 and Kcnh2 have a much wider expression profile than strictly confined to the myocardium, similar to their putative regulatory Kcne1-5 beta-subunits. At present, the distribution of distinct potassium channel subunits has been partially mapped in adult tissues, whereas almost no information is available during embryonic development. In this study, we report a detailed analysis of Kcnq1, Kcnh2, and Kcne3 protein expression during mouse embryogenesis. Our results demonstrate that Kcnq1 and Kcnh2 are widely distributed. Coexpression of both alpha-subunits is observed in a wide variety of organs, such as heart and the skeletal muscle, whereas others display unique Kcnq1 or Knch2 expression. Interestingly, Kcne3 expression is also widely observed in distinct tissue layers during embryogenesis, supporting the notion that an exquisite balance of alpha- and beta-subunit expression is required for modulating potassium conductance in distinct organs and tissue layers.

Animals↗

Lymphocyte commitment during embryonic development, in the mouse.

Multipotent hematopoietic stem cells (HSC) differentiate into mature cells in the fetal liver (FL) during embryonic development, and in the bone marrow (BM) in adult animals. Multilineage differentiation is accomplished by the stepwise commitment of stem cells that sequentially loose differentiation potential. The characterization of the intermediate lymphoid precursors isolated from both hematopoietic sites suggests that, in FL, their potential of differentiation as well as their growth factor requirements are apparently less strict than in the BM. This could be the result of different commitment strategies at those sites: stochastic in the FL and instructive in the BM.

Animals↗

Embryonic development of the Drosophila brain: formation of commissural and descending pathways.

The establishment of initial axonal pathways in the embryonic brain of Drosophila melanogaster was investigated at the cellular and molecular level using antibody probes, enhancer detector strains and axonal pathfinding mutants. During embryogenesis, two bilaterally symmetrical cephalic neurogenic regions form, which are initially separated from each other and from the ventral nerve cord. The brain commissure that interconnects the two brain hemispheres is pioneered by axons that project towards the midline in close association with an interhemispheric cellular bridge. The descending longitudinal pathways that interconnect the brain to the ventral nerve cord are prefigured by a chain of longitudinal glial cells and a cellular bridge between brain and subesophageal ganglion; pioneering descending and ascending neurons grow in close association with these structures. The formation of the embryonic commissural and longitudinal pathways is dependent on cells of the CNS midline. Mutations in the commissureless gene, which affects growth cone guidance towards the midline, result in a marked reduction of the brain commissure. Mutations in the single-minded gene and in other spitz group genes, which affect the differentiation of CNS midline cells, result in the absence or aberrant projection of longitudinal pathways. The analysis of axon pathway formation presented here reveals remarkable similarities as well as distinct differences in the embryonic development of the brain and the segmental ganglia, and forms the basis for a comprehensive genetic and molecular genetic dissection of axonal pathfinding processes in the developing brain.

Animals↗

Melanoma Cell Adhesion Molecule (MCAM) expression in the myogenic lineage during early chick embryonic development.

We describe the expression pattern of cMCAM, a cell adhesion molecule of the immunoglobulin superfamily, in early chick embryonic development by in situ hybridisation. An initial ectodermal domain of expression is subsequently expanded, and cMCAM is expressed in the neural crest cells, otic vesicle, heart primordium, notochord and endoderm. In addition, cMCAM expression localises in the myotome once the somite cells have been specified. An in vitro murine cellular system allowed us to confirm that MCAM expression coincides with the onset of myogenic cell determination.

Alternative Splicing↗

The human sperm centrosome is responsible for normal syngamy and early embryonic development.

As early as 1887, it was postulated that the mature oocyte possesses all of the elements necessary for embryonic development with the exception of an active division centre, and that the spermatozoon contains such a centre, but lacks the substrate in which to operate. This division centre is called the centrosome. The precise definition of this structure is still a subject for debate. It consists of two centrioles in a perpendicular arrangement and pericentriolar material, and is considered to be responsible for nucleation of microtubules and the formation of the mitotic spindle. There is a paternal pattern of inheritance of the centrosome in humans; thus, human oocytes lack centrioles but the spermatozoa carry two. At gamete fusion the sperm tail is incorporated into the ooplasm, and the centriolar region forms the sperm aster while the sperm head is decondensing; this aster acts to guide the female pronucleus towards the male pronucleus. The centriole duplicates during the pronuclear stage, and at syngamy centrioles are found at opposite poles of the first cleavage. The centrosome has several implications for human infertility. It is possible that immotile or nonprogressively motile spermatozoa may possess centriolar abnormalities or an absence of centrioles. Similarly, antisperm antibodies against centrioles may be responsible for mitotic arrest. One way of solving this problem would be the use of donor centrosomes. To this end, we have assessed the ability of embryos injected with physically separated sperm segments (head only, head and tail separated or isolated tail) to develop normally. Fluorescent in situ hybridization revealed an almost universal mosaicism in these embryos, suggesting that physical disruption of the spermatozoa compromises the ability of the centrosome to function in the zygote. Thus far, centrosome donation with centriole-carrier flagellae obtained by this dissection method does not appear to be feasible.

Cell Cycle↗

Expression of DRG during murine embryonic development.

We had previously characterised a cDNA which encodes a novel GTP-binding protein DRG. The expression of drg gene is down-regulated during the embryonic development of murine central nervous system. Further analysis of drg mRNA and protein in adult mouse tissues and various cell lines of different origins indicated that it is expressed widely, albeit at low and variable levels. In situ hybridisation analysis of mRNA expression in sections of mouse embryos indicated that drg is expressed strongly in various embryonic tissues. The expression of drg mRNA is greatly reduced in newborn animals. At cellular level, DRG protein can be detected in the cytoplasm. These observations suggest that DRG may play multiple roles in development and normal cell metabolism.

3T3 Cells↗

Generalized abnormal embryonic development in missed abortion: embryoscopic and cytogenetic findings.

A direct view of the embryo by means of transcervical embryoscopy prior to evacuation in 154 cases of missed abortion showed general embryonic maldevelopment in 48 cases (31%). A successful cytogenetic evaluation of these growth-disorganized embryos was performed in 37. Chromosomal abnormalities were found in 26 cases (70%), with autosomal trisomies in 24 cases (92%). Trisomies involved chromosome 3 (one case), 6 (one case), 8 (two cases), 10 (one case), 12 (two cases), 14 (one case), 16 (11 cases), 20 (one case), and 22 (four cases). Most of these chromosome abnormalities represented nonviable defects, and their presence explained the minimal embryonic development observed embryoscopically. An apparently normal karyotype was observed in 11 growth-disorganized embryos whose maldevelopment was similar to that resulting from the trisomies listed above. The factors responsible for embryonic maldevelopment with a normal karyotype are presently unknown and require further study, including investigation of imprinting defects, subtelomeric abnormalities, and cryptic mosaicism.

Abortion, Missed↗

Embryonic development and mitochondrial function. 2. Thiamphenicol induced embryotoxicity.

Inhibition of mitochondrial protein synthesis in rat embryos during late organogenesis leads to impaired embryonic development. 1. Thiamphenicol (TAP), similar to chloramphenicol, inhibits in vivo the synthesis of cytochrome oxidase (cytox), which is partially synthesized by the mitochondrion. Subsequently, DNA synthesis and embryonic growth are affected. 2. Embryos on day 10 and 11, in contrast to embryos on day 9 of gestation, show a high sensitivity of mitochondrial protein synthesis, measured as cytox activity. From day 10 onwards, such an inhibition leads to pronounced impairment of DNA synthesis. The rat hemochorial placenta starts functioning on day 12 of gestation. Larger doses of TAP are required to inhibit cytox and DNA synthesis for treatment after placentation rather than before placentation. 3. Dose-response relationships differ depending on the date and duration of treatment. Application of TAP for 1 day requires 10-30 mg/kg TAP to inhibit cytox synthesis and 60-100 mg/kg to impair embryonic growth. Prolongation of treatment to 4 days (day 10-13) lowers the dose required for inhibition of DNA synthesis to 10 mg TAP/kg/day. This is lower than the human therapeutic dose. Larger doses lead to embryolethality. 4. The extent of inhibition of DNA synthesis provoked by inhibition of mitochondrial protein synthesis depends on a number of factors which include: different growth rates during organogenesis, the number of mitochondria present prior to treatment, availability of extramitochondrial ATP sources and placental permeability barrier.

Animals↗

RAR beta isoforms: distinct transcriptional control by retinoic acid and specific spatial patterns of promoter activity during mouse embryonic development.

That both deficiency and excess of vitamin A lead to a wide spectrum of congenital abnormalities has strongly implicated the active metabolite, retinoic acid (RA), in normal embryonic development. There are 3 families of RA receptors (RARs), RAR alpha, RAR beta and RAR gamma, each having at least two isoforms derived from primary transcripts initiated at two promoters P1 and P2 (reviewed in Leid et al., 1992) Transcripts encoding all 4 isoforms of RAR beta (RAR beta 1 to RAR beta 4) accumulate in embryonal carcinoma (EC) cells in the presence of RA. It has been previously shown that the RA modulation of RAR beta 2/beta 4 transcripts is achieved at the level of transcriptional initiation via a RA response element (RARE) present in the P2 RAR beta 2/beta 4 promoter. In contrast, the mechanism by which RA up-regulates RAR beta 1/beta 3 transcripts has not yet been elucidated. We describe here the isolation of the P1 RAR beta 1/beta 3 promoter and characterization of its activity in transgenic animals. We find that RAR beta 1/beta 3 promoter activity, which is apparently confined to the embryonic CNS, is not modified by RA treatment, unlike that of the RAR beta 2/beta 4 promoter (Mendelsohn et al., 1991). Nuclear run-on transcription analysis in EC cells supports the conclusion that RAR beta 1/beta 3 transcript initiation is not modulated by RA, and that the RA-induced accumulation of RAR beta 1/beta 3 transcripts occur via a RA-dependent release of a block in RNA chain elongation.

Animals↗

Mouse zinc transporter 1 gene provides an essential function during early embryonic development.

The SLC30 family of cation diffusion transporters includes at least nine members in mammals, most of which have been documented to play a role in zinc transport. The founding member of this family, Znt1, was discovered by virtue of its ability to efflux zinc from cells and to protect them from zinc toxicity. However, its physiological functions remain unknown. To address this issue, mice with targeted knockout of the Znt1 gene were generated by homologous recombination in embryonic stem cells. Heterozygous Znt1 mice were viable. In contrast, homozygous Znt1 mice died in utero soon after implantation due to a catastrophic failure of embryonic development. Although extraembryonic membranes formed around these embryos, the embryo proper failed to undergo morphogenesis past the egg cylinder stage and was amorphous by d9 of pregnancy. Expression of the Znt1 gene was detected predominantly in trophoblasts and in the maternal deciduum during the postimplantation period (d5 to d8). The failure of homozygous Znt1 embryos to develop could not be rescued by manipulating maternal dietary zinc (either excess or deficiency) during pregnancy. However, embryos in Znt1 heterozygous females were approximately 3 times more likely to develop abnormally when exposed to maternal dietary zinc deficiency during later pregnancy than were those in wildtype females. These studies suggest that Znt1 serves an essential function of transporting maternal zinc into the embryonic environment during the egg cylinder stage of development, and further suggest that Znt1 plays a role in zinc homeostasis in adult mice.

Animals↗

Adenylic nucleotides and energy charge during the embryonic development of Bufo arenarum.

The contents of ATP, ADP and AMP were determined by HPLC and adenylic energy charge (AEC) was estimated during different stages of the embryonic development of Bufo arenarum up to the tailbud stage. All the developmental stages studied showed a high ATP content (about 1.04-1.48 nmol/emb.). The concentration of ADP was low (0.025-0.041 nmol/emb.) but rose slightly at the neural tube stage. AMP was undetectable before the tailbud stage. AEC values were almost constant (about 0.987-0.992) throughout the period studied. Only a fall at the tailbud stage could be detected which can be related to this more advanced cellular differentiation stage.

Adenine Nucleotides↗

GLUT8 is dispensable for embryonic development but influences hippocampal neurogenesis and heart function.

GLUT8 is a glucose transporter isoform expressed at high levels in testis; at intermediate levels in the brain, including the hippocampus; and at lower levels in the heart and several other tissues. GLUT8 is located in an intracellular compartment and does not appear to translocate to the cell surface, except in blastocysts, where insulin has been reported to induce its surface expression. Here, we generated mice with inactivation of the glut8 gene. We showed that expression of GLUT8 was not required for normal embryonic development and that glut8-/- mice had normal postnatal development, glucose homeostasis, and response to mild stress. Adult glut8-/- mice showed increased proliferation of hippocampal cells but no defect in memory acquisition and retention. Absence of GLUT8 from the heart did not alter heart size and morphology but led to an increase in P-wave duration, which was not associated with abnormal Nav1.5 Na+ channel or connexin expression. Thus, absence of GLUT8 expression in the mouse caused complex but mild physiological alterations.

Animals↗

Characterization of Drosophila Rad51/SpnA protein in DNA binding and embryonic development.

The Rad51 is a highly conserved protein throughout the eukaryotic kingdom and an essential enzyme in DNA repair and recombination. It possesses DNA binding activity and ATPase activity, and interacts with meiotic chromosomes during prophase I of meiosis. Drosophila Rad51, Spindle-A (SpnA) protein has been shown to be involved in repair of DNA damage in somatic cells and meiotic recombination in female germ cells. In this study, DNA binding activity of SpnA is demonstrated by both agarose gel mobility shift assay and restriction enzyme protection assay. SpnA is also shown to interact with meiotic chromosomes during prophase I in the primary spermatocytes of hsp26-spnA transgenic flies. In addition, SpnA is highly expressed in embryos, and the depletion of SpnA by RNA interference (RNAi) leads to embryonic lethality implying that SpnA is involved in early embryonic development. Therefore, these results suggest that Drosophila SpnA protein possesses properties similar to mammalian Rad51 homologs.

Animals↗

Blood vessel/epicardial substance (bves) expression, essential for embryonic development, is down regulated by Grk/EFGR signalling.

The Pop1/Bves (blood vessel/epicardial substance) gene is a member of the popeye gene family recently identified in various species. It encodes a potential transmembrane glycoprotein and is a cell adhesion molecule present in skeletal and cardiac muscle and epithelia. We isolated the Drosophila homologue of Bves (DmBves) and found, using in situ hybridisation to RNA in ovaries, that bves is expressed in all follicular epithelial cells surrounding the oocyte at stage 10, except those in very posterior and anterior-dorsal regions adjacent to the oocyte. We show that the repression of bves expression in anterior-dorsal follicle cells is regulated by the Grk/EGFR signalling pathway. Bves is also expressed in nurse cells during oogenesis and its transcripts are then translocated into the oocyte. Expression of bves antisense RNA during oogenesis causes reduced viability in the resulting embryos. There is a failure in the migration of pole cells from the posterior towards the antero-dorsal side of the embryo, probably resulting from abnormal germband extension and we suggest that bves is essential for normal embryonic development.

Amino Acid Sequence↗