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PubMed2005

Overexpression of connexin43 alters the mutant phenotype of midgestational wnt-1 null mice resulting in recovery of the midbrain and cerebellum.

The midbrain-hindbrain (MHB) junction plays a key role in the patterning of the embryonic neural tube and the formation of brain structures such as the cerebellum. The mitogen wnt-1 is critical for cerebellar development, as evidenced by the lack of MHB region and cerebellar formation in the wnt-1 null embryo. We have generated wnt-1 null embryos overexpressing the gap junction gene connexin43 by crossing wnt-1 null heterozygotes into the CMV43 mouse line. We have confirmed that these mice show an increase in gap junctional communication by dye coupling analysis. Two-thirds of wnt-1 null CMV43(+) mouse embryos at E18.5 have a cerebellum. In addition, changes in the wnt-1 null phenotype in mouse embryos overexpressing connexin43 are observed as early as E9.5. At this stage, one-quarter of wnt-1 null CMV43(+) embryos display extra or expanded tissue present at the MHB boundary (a wnt-1 null enlarged phenotype). In situ hybridization studies conducted on these embryos have indicated no changes in the expression of embryonic brain positional markers in this region. We conclude from these studies that overexpression of the connexin43 gap junction restores cerebellar formation by compensating for the loss of wnt-1.

Animals↗
PubMed2002

Pluripotent cell division cycles are driven by ectopic Cdk2, cyclin A/E and E2F activities.

Pluripotent cells of embryonic origin proliferate at unusually rapid rates and have a characteristic cell cycle structure with truncated gap phases. To define the molecular basis for this we have characterized the cell cycle control of murine embryonic stem cells and early primitive ectoderm-like cells. These cells display precocious Cdk2, cyclin A and cyclin E kinase activities that are conspicuously cell cycle independent. Suppression of Cdk2 activity significantly decreased cycling times of pluripotent cells, indicating it to be rate-limiting for rapid cell division, although this had no impact on cell cycle structure and the establishment of extended gap phases. Cdc2-cyclin B was the only Cdk activity that was identified to be cell cycle regulated in pluripotent cells. Cell cycle regulation of cyclin B levels and Y(15) regulation of Cdc2 contribute to the temporal changes in Cdc2-cyclin B activity. E2F target genes are constitutively active throughout the cell cycle, reflecting the low activity of pocket proteins such as p107 and pRb and constitutive activity of pRb-kinases. These results show that rapid cell division cycles in primitive cells of embryonic origin are driven by extreme levels of Cdk activity that lack normal cell cycle periodicity.

Animals↗
PubMed1997

The elusive search for fetal infection. Changing the gold standards.

Two percent to four percent of all newborns have a major structural malformation of which only a small percentage can be explained by either aneuploidy or a single-gene disorder. No other mammal has a similar reproductive problem. It is the suggestion that previously unrecognized viral infection accounts for a large percentage of these abnormalities by interfering with appropriate embryonic cell migration, reducing hyperplasia, or damaging the precursor structure by means of an inflammatory mechanism.

Algorithms↗
PubMed1991

Isolation and Characterization of 51 embryo-specific Mutations of Maize.

A plant embryo consists of an embryonic axis, which eventually grows into the adult body, and one or two nutritive structures, the cotyledons. In the grasses embryo morphogenesis can be divided into three periods: during the first the embryo is regionalized into an embryo proper and suspensor, during the second the embryonic axis is established, and during the third vegetative structures are elaborated. Maize, with its well-characterized embryo-genesis, powerful genetics, and transposon tagging stocks, offers an attractive system for mutational analysis of these events. We have isolated 51 embryo-specific (emb) mutations from active Robertson's Mutator maize stocks. These are single-gene recessive lethals that represent at least 45 independent mutation events. Each of the 25 mutations was located to a chromosome arm using a B-A translocation set that uncovers approximately 40% of the genome; the same test failed to locate 20 others. The embryo phenotype of 27 mutations was characterized by examining mature mutant embryos in fresh dissection: the various emb mutations differ in phenotype and each is consistent in its expression. All 27 mutations result in retarded embryos that are morphologically abnormal. Nine mutants are blocked during the first period; 10 mutants are blocked during the second period; and eight mutants are blocked during the third period. Based on both the genetic and developmental data, it is likely that there are many loci that can mutate to give the emb phenotype and that these genes are crucial to the morphogenesis of the embryo.

Journal Article↗
PubMed2005

A cytokine secreted from the suboesophageal body is essential for morphogenesis of the insect head.

The suboesophageal body of insects was identified over a century ago in the silkworm embryo, but its biological function is still unknown. We discovered that this tissue is differentiated in the earliest embryonic stages of the cabbage armyworm and secretes the insect cytokine, growth-blocking peptide (GBP), transiently from 24 to 60 h after oviposition when gastrulation is in progress. Over-expression of GBP, achieved by microinjection of the GBP gene driven by a cytomegalovirus (CMV) constitutive promoter, resulted in complex deformities of the procephalon (embryonic head). Severe abnormal phenotypes of the head structure were produced by silencing the GBP expression in the embryo by treating with GBP double-stranded RNA: the procephalon-containing optic lobes diminished and completely separated into bilateral halves. This indicates that GBP secreted from the suboesophageal body plays an essential role in the formation of the procephalic domain during early embryogenesis. The cytokine-induced fusion of bilateral procephalic lobes is thought to be evolutionarily conserved at least in insects, because of the widespread occurrence of the suboesophageal body in insect embryos.

Animals↗
PubMed1987

[Homotopic transplantation of embryonic neocortical tissue into the brain of adult rats after its damage].

Structural characteristics (survival, growth, connections) have been studied in the transplant of the cerebral cortex tissue in Wistar rat embryos (18-day-old), implanted into the brain of mature rats of the same line at various time after a partial lesion of the sensomotor cortex. In 3-5 months after transplantation the light microscopy methods demonstrate that spatial interconnections of the transplant and the injured brain of the recipient depend on time interval between the cerebral lesion and transplantation of the embryonal nervous tissue. Horseradish peroxidase (HP) is ionophoretically injected into the recipient's cerebral tissue away from the place of transplantation. In the transplant retrogradely labelled HP neurons are revealed. This demonstrates efferent connections of the implanted tissue with the host's brain. Presence of the anterogradely labelled nervous terminals in the transplant tissue demonstrates existence of afferent connections of the transplant with the recipient's tissue. Possible mechanisms of survival, growth and formation of connections of the transplant in the injured brain of the mature animal are discussed.

Animals↗
PubMed1986

Chromatin structural transitions and the phenomenon of vitellogenin gene memory in chickens.

We have previously shown that the steroid hormone-mediated transcriptional activation of the chicken vitellogenin II gene (VTGII) in the liver is accompanied by a series of chromatin structural changes, including the formation of two sets of 5'-proximal nuclease-hypersensitive sites and the demethylation of a single 5'-flanking MspI site which lies within a region of DNA that recently has been shown by Jost and co-workers to specifically bind the estrogen receptor complex in vitro. To assay the stability and possible functional significance of these induced structural changes, we transiently activated the VTGII gene during embryonic development and then allowed the chickens to hatch and grow for various periods of time before analyzing their livers. By 7 weeks posthatching all of the induced 5'-flanking hypersensitive sites had decayed. Moreover, the loss of these sites occurred without consequence to the "memory effect," that is, these structural features did not need to be present in hormone withdrawn birds to allow this gene to be activated more rapidly in response to a secondary presentation of estradiol. Although the demethylation was more stable, it also appeared not to be the basis of the memory phenomenon. The birds that still exhibited memory after 25 weeks of hormone withdrawal were not more extensively demethylated within the receptor-binding site than were the birds which failed to show memory at this age. A similar uncoupling of these two parameters was also observed when embryos were first injected with submaximal doses of estradiol and then assayed 1 week after hatching; the chickens which acquired memory were not demethylated to any greater extent than those which did not acquire memory. Other parameters that may be relevant to memory are discussed.

Animals↗
PubMed2005

Research of blastocyte-like structure in chicken.

The chicken embryo is a classic model used to investigate embryonic development, gene expression, and tissue differentiation, and is also an important research tool in studying the animal functional genomics. The whole blastoderms of fresh unincubated eggs from White Leghorn chickens were collected with a paper ring, mechanically broken into small pieces and cultured in medium. Then the small pieces would develop into blastocyte-like structures (BLS), which could be facilitated by an addition of fetal bovine serum (FBS) to the primary culture and their diameter was nearly doubled from 12 to 24 h. The additional yolk had no positive effect on the development in the first 12 h but encouraged the BLSs attaching and inner cells differentiating instead in 24 h. The inner cells of the BLS showing a high alkaline-phosphatase activity similar to those in mouse embryonic stem (ES) cells and also expressing a large amount of the specific stage embryonic antigen-1 (SSEA-1) on the surface, which was known to be the characteristic of non-differentiated mouse and avian ES cells, could finally differentiate into nerve-like cells, fibroblast cells and so on in the medium. Leukemia inhibitory factor (LIF) facilitated the cells' proliferation and prevented differentiation in the suspended culture of the BLSs. So we drew the conclusion that the BLS obtained from broken blastoderm can be used to amplify avian ES cells so as to initiate a new method of producing transgenic chickens.

Animals↗
PubMed1985

Developmental changes in internal structure of chick heart plasma membrane.

Although changes in electrophysiologically measurable membrane properties of chick embryo cardiac plasma membrane have been repeatedly documented during embryonic development, ultrastructural techniques were heretofore too insensitive to detect developmental changes in internal structure of this membrane. We report here significant structural changes detected by applying the quantitative analysis of Kordylewski, Karrison, and Page (Amer. J. Physiol. 245, H992-H997, 1983 and 248, H297-H304, 1985) to stereo imaged electron microscopic negatives of glutaraldehyde-fixed chick embryo hearts, freeze fractured and photographed with a goniometer stage. Between Hamburger-Hamilton stages 12+ (about 48 hr incubation) and 24 (about 96 hr incubation), plasmalemmal P-face particle density of ventricular myocytes increased from 2228 +/- 139 to 3063 +/- 109 (P less than 0.01); thereafter, measurements at stages 30, 37, 40, and 45 (7, 11, 15, and 19 days incubation) showed a slower significant linear increase which gave a least-squares line with a slope of 41 +/- 13 particles/day (P less than 0.01). Just before hatching, (stage 45) the value of 3762 +/- 234 was similar to, though slightly smaller than, the values of 4122 +/- 153 (8 days after hatching) and 4281 +/- 218 (adult chicken). These results indicate striking stage-dependent changes in the population of integral membrane proteins (channels, carriers, receptors, etc.), especially marked during early embryogenesis.

Animals↗
PubMed1999

Turnover of tubulin in ciliary outer doublet microtubules.

Previous pulse-chase labeling studies have shown that structural proteins incorporate into fully assembled sea urchin embryonic cilia at rates approaching those of full regeneration. When all background ciliogenesis was suppressed by taxol, the turnover of most proteins, including tubulin, continued. The present study utilized chemical dissection to explore the route of tubulin incorporation in the presence of taxol and also in steady-state cilia from prism stage embryos. Surprisingly, in cilia from untreated embryos, the most heavily labeled tubulin was found in the most stable portion of the doublet microtubles, the junctional protofilaments. With taxol, this preferential incorporation was suppressed, although control-level turnover still took place in the remainder of the doublet. This paradoxical result was confirmed by pulse-chase labeling and immediately isolating steady-state cilia, then isolating two additional crops of cilia regenerated, respectively, from pools of high and then decreased label. In each case, the level of label occurring in the tubulin from the junctional protofilaments, compared with that from the remainder of the doublet, correlated with the level of pool label from which it must exchange or assemble. These data indicate that ciliary outer doublet microtubules are dynamic structures and that the junctional region is not inert. Plausible mechanisms of incorporation and turnover of tubulin in fully-assembled, fully-motile cilia can now be assessed with regared to recent discoveries, particularly intraflagellar transport, distal tip incorporation, and treadmilling.

Animals↗
PubMed2003

Roles of NPM2 in chromatin and nucleolar organization in oocytes and embryos.

Upon fertilization, remodeling of condensed maternal and paternal gamete DNA occurs to form the diploid genome. In Xenopus laevis, nucleoplasmin 2 (NPM2) decondenses sperm DNA in vitro. To study chromatin remodeling in vivo, we isolated mammalian NPM2 orthologs. Mouse NPM2 accumulates in oocyte nuclei and persists in preimplantation embryos. Npm2 knockout females have fertility defects owing to failed preimplantation embryo development. Although sperm DNA decondensation proceeds without NPM2, abnormalities are evident in oocyte and early embryonic nuclei. These defects include an absence of coalesced nucleolar structures and loss of heterochromatin and deacetylated histone H3 that normally circumscribe nucleoli in oocytes and early embryos, respectively. Thus, Npm2 is a maternal effect gene critical for nuclear and nucleolar organization and embryonic development.

Acetylation↗
PubMed1979

[Electron microscopic study of the mitochondria in the apical meristem of a wheet shoot in ontogeny].

The mitochondria of apical meristem cells in the wheat (Triticum aestivum L.) shoot were studied during ontogenesis using electron microscope and morphometrical methods. Changes in their structure were followed from the juvenile mitochondria of the seed embryonic ear cells. The parameters of the "average" mitochondrion, such as profile area, outer membrane length, were shown to differ relatively weakly during the periods with different meristem activity. Changes in the internal structure of the mitochondria having the developed system of crystae in the actively growing apices and those with weakly developed crystae in the resting seed or low active "waiting meristem" are much more pronounced. The relative volume of mitochondria, their number per unit of cytoplasm volume and total length of membranes suffer relatively insignificant changes during the vegetative phase and increase markedly during the prefloral phase when the apex is preparing itself for generative differentiation.

Microscopy, Electron↗
PubMed1987

Significance of carbohydrate components of cell surfaces.

This article is focused on a family of carbohydrate structures which are (a) target antigens of autoantibodies and (b) onco-developmental antigens which change during embryonic development, cell differentiation, maturation and oncogenesis. Among the carrier molecules of these saccharide structures is the receptor for epidermal growth factor. Perturbation of these structures on the isolated receptor enhances autophosphorylation of the receptor glycoprotein. This suggests that the carbohydrate chains may be part of a growth regulatory network which may be 'tuned' or perturbed via interactions with endogenous lectins or by adhesins of infective agents. Certain sialylated forms of these oligosaccharide structures serve as receptors for a pathogen of man, Mycoplasma pneumoniae which, following infection, elicits anti-erythrocyte autoantibodies. These autoantibodies are directed against the backbone domain of the carbohydrate receptor and are therefore anti-receptor antibodies. These observations suggest that complex formation between the adhesins of infective agents and specific saccharides of host-cell membranes may be a 'new' mechanism for eliciting autoantibodies.

Antigens, Surface↗
PubMed2003

Ultrastructure of the ovarian germ cells in the quail embryos, with special reference to the oocytes.

Developing germ cells from oogonia to oocyte stages in embryonic quail ovaries were observed ultrastructurally. In contrast with those of chicks, quail oocytes showed some conspicuous structures. Typical feature of the Balbiani body, which was constructed by eccentrical concentration of whole organelles, was observed through the oocyte stage, although loose aggregations of organelles were found even at the oogonial stage. Such striking feature of Balbiani body was not observed as yet in any animal species. Golgi apparatus, which occupied the center of a Balbiani body, was often found attaching with the nuclear membrane. This finding suggests that the Balbiani body play some role for the oocyte maturation. Upon the nuclear membrane of oocytes, a great number of electron dense structures were found. They were thought to be a kind of nuclear pore complexes. Such abundance of the nuclear pore complexes was another structural characteristic of quail oocytes. They increased in number according to the progress of embryonic development, although small numbers were also seen even at the oogonial stage.

Animals↗
PubMed1991

Stromal assemblies containing collagen types IV and VI and fibronectin in the developing embryonic avian cornea.

The morphogenesis of type IV collagen-containing structures in the stromal matrix of the developing avian cornea was investigated using immunofluorescence and immunoelectron microscopic histochemistry. Two forms of type IV collagen-containing structures were seen; these differed in their probable origin, structure, molecular composition, and developmental fate. The major form of stromal type IV collagen-containing material, termed "strings," was observed only after swelling of the primary stroma and the onset of mesenchymal invasion. These strings are presumed to be products of the stromal cells. In immunofluorescence histochemistry they appeared as linear segments of type IV collagen-specific immunoreactivity. In immunoelectron microscopy, they appeared initially as electron-dense sausages of variable length and orientation. They frequently were associated with cell surfaces and, in fortuitous sections, appeared to connect adjacent cells. The strings also contained type VI collagen and fibronectin, but very little, if any, of the basement membrane components laminin and heparin sulfate proteoglycan (HSPG). As the stroma continued to expand in thickness, more of these structures were observed in a radial orientation, becoming quite long and less tortuous. Later in development, as stromal condensation proceeded, they disappeared. We suggest that the strings function to stabilize the stromal matrix, and perhaps to limit the rate and/or extent of stromal expansion, during a phase of rapid swelling and matrix deposition. The other form of type IV collagen-containing stromal material appeared as irregularly shaped plaques of basement membrane-like material identical to those previously described in mature corneas. These are likely derived from the corneal endothelial cells. They contained other basement membrane-associated components (laminin, HSPG) and fibronectin, but not type VI collagen. This material persists in mature corneas as sparse irregular stromal plaques and as matrix in the interface between Descemet's membrane and the corneal stroma.

Age Factors↗
PubMed1990

The mouse keratin 19-encoding gene: sequence, structure and chromosomal assignment.

Keratin 19 (K19) is synthesized mainly in embryonic and adult simple epithelia, but has also been found in stratified epithelia as well. K19 is the smallest known keratin and is remarkable in that, contrary to all other keratins, it does not have a designated partner for the formation of filaments, implying that regulation of its expression is different from other keratin-encoding genes. As a first step in elucidating the mechanisms by which the K19 gene is regulated in relatively undifferentiated embryonic and in terminally differentiated adult tissues, a series of overlapping clones containing the complete mouse K19 gene was isolated from a mouse genomic library and characterized. The nucleotide (nt) sequence extends over 5119 nt and includes six exons. A region of 303 nt upstream from the transcription start point (tsp) was also sequenced. Comparison with the human and bovine K19 genes revealed the existence of homologies in both the coding and noncoding regions. The putative promoter region of the mouse K19 gene is highly homologous to the corresponding sequences of the human and bovine K19 genes. It contains an ATA box, a CAAT box and two potential Sp1-binding sites. Significant homologies were also found between the sequences of the introns of the mouse, human and bovine genes: this was particularly evident in introns 2, 3, 4 and 5. Intron 1, which showed the greatest degree of divergence, was found to contain many repetitive elements. Finally, it is shown that the mouse K19 gene cosegregates with the type-I keratin-encoding gene locus (Krt-1) on chromosome 11.

Amino Acid Sequence↗
PubMed2006

Molecular markers of neuronal progenitors in the embryonic cerebellar anlage.

The cerebellum, like the cerebrum, includes a nuclear structure and an overlying cortical structure. Experiments in the past decade have expanded knowledge beyond the traditional function of the cerebellum to include critical roles in motor learning and memory and sensory discrimination. The initial steps in cerebellar development depend on inductive signaling involving FGF and Wnt proteins produced at the mesencephalic/metencephalic boundary. To address the issue of how individual cerebellar cell fates within the cerebellar territory are specified, we examined the expression of transcription factors, including mammalian homologues of LIM homeodomain-containing proteins, basic helix-loop-helix proteins, and three amino acid loop-containing proteins. The results of these studies show that combinatorial codes of transcription factors define precursors of the cerebellar nuclei, and both Purkinje cells and granule neurons of the cerebellar cortex. Examination of gene expression patterns in several hundred lines of Egfp-BAC (bacterial artificial chromosome) transgenic mice in the GENSAT Project revealed numerous genes with restricted expression in cerebellar progenitor populations, including genes specific for cerebellar nuclear precursors and Purkinje cell precursors. In addition, we identified patterns of gene expression that link granule and Purkinje cells to their precerebellar nuclei. These results identify molecular pathways that offer new insights on the development of the nuclear and cortical structures of the cerebellum, as well as components of the cerebellar circuitry.

Animals↗
PubMed2002

[Conditional targeting of p16(INK4a)exon 1a in mouse embryonic stem cells].

OBJECTIVE: To study the relationship between targeting vector structure and homologous recombination rate and investigate whether the mouse p16(INK4a) plays a role in tumor suppression. METHODS: A conditional targeting vector with 2.0 kb EcoR I/Xba I fragment as short arm and 5.9 kb SpeI/NotI fragment as long arm was built. Of the 2 direct locus crossing- over(loxPs) in the vector, one was inserted at 240 bp upstream of the initiate code of p16(INK4a) exon 1a and the other at 1633 bp downstream of the initiate code. Both exon 1a and the selection marker Neo will be deleted in targeted cells when mediated by Cre. After linearlization and purification, t he targeting vector was introduced into ES cells through electroporation. RESULTS: Twenty-four G418- and gancyclovir-resistant ES cell colonies were picked out and one of them was confirmed as positive by Southern hybridization. CONCLUSION: Targeting vectors with 2 TK genes flanking the homologous arms are likely to produce good result of homologous recombination.

Animals↗