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Identification of potential target genes for RFX4_v3, a transcription factor critical for brain development.

Regulatory factor X4 variant transcript 3 (Rfx4_v3) gene disruption in mice demonstrated that interruption of a single allele (heterozygous, +/-) prevented formation of the subcommissural organ, resulting in congenital hydrocephalus, while interruption of two alleles (homozygous, -/-) caused fatal failure of dorsal midline brain structure formation. To identify potential target genes for RFX4_v3, we used microarray analysis to identify differentially expressed genes in Rfx4_v3-deficient mouse brains at embryonic day 10.5, before gross structural changes were apparent. Of 109 differentially expressed transcripts, 24 were chosen for validation and 22 were confirmed by real-time PCR. Many validated genes encoded critical proteins involved in brain morphogenesis, such as the signaling components in the Wnt, bone morphogenetic protein (BMP) and retinoic acid (RA) pathways. Cx3cl1, a CX3C-type chemokine gene that is highly expressed in brain, was down-regulated in the Rfx4_v3-null mice. Both human and mouse Cx3cl1 proximal promoters contained highly conserved X-boxes, known cis-acting elements for RFX protein binding. Using the Cx3cl1 promoter as an example of a target gene, we demonstrated direct binding of RFX4_v3 to the Cx3cl1 promoter, and trans-acting activity of RFX4_v3 protein to stimulate gene expression. These data suggest that RFX4_v3 may act upstream of critical signaling pathways in the process of brain development.

Animals↗

Noggin and basic FGF were implicated in forebrain fate and caudal fate, respectively, of the neural tube-like structures emerging in mouse ES cell culture.

We developed neural tube-like structures accompanying neural crest-like cells by treating embryonic stem (ES) cells with retinoic acid. The structures contained pseudostratified Nestin+Vimentin+ neuroepithelial cells surrounded by Masson staining+ basement membrane. betaIIItubulin+Synaptophysin+ mature neurons and glial fibrillary acidic protein (GFAP)+ glial cells dispersed outside of the membrane. Addition of Noggin to the culture induced prominent proliferation of the neuroepithelial cells, leading to epithelial hyperstratification of the structures. mRNAs of transcription factors essential for forebrain development such as Emx1/2 and Pax6 were specifically expressed and Islet1+Lim1/2- motoneurons appeared by the addition of Noggin. In contrast, basic fibroblast growth factor (bFGF) promoted enlargement of central lumen and elongation of the structures. mRNAs of caudal markers, Gbx2, Cdx2 and Hoxb4/9 were expressed and Lim1/2+ spinal motoneurons appeared by the addition of bFGF. Addition of BMP-4 similarly brought about mild enlargement of central lumen of the structures. Interestingly, the addition of BMP-4 induced Slug+ neural crest-like cells surrounding the tube-like structures. mRNAs of Snail and dHand, other markers for neural crest cells, were also expressed by the addition of BMP-4. These results suggest that Noggin lead the neural-tube like structures to forebrain fate, whereas bFGF was involved in the caudalization. BMP-4 was implicated in emergence of the neural crest-like cells. Differentiation of ES cells by the present methods may mimic neurulation and subsequent neural development of early embryos, and elucidates the opposite effects of Noggin and bFGF for the neural tube development.

Animals↗

High idiotypic connectivity of the VH7183-encoded antibodies directed to a murine embryonic carbohydrate antigen, Lewis Y, as ascertained by syngenic anti-idiotype monoclonal antibodies.

The Lewis Y Ag is a carbohydrate Ag which is closely related to a well-known murine embryonic Ag, the stage-specific embryonic Ag-1 (SSEA-1), in its biochemical structure. It is expressed at the surface of murine embryonic cells as well as many murine cancer cells. For the analysis of idiotopes carried by the anti-Lewis Y antibodies, we generated two syngenic anti-idiotypic mAb, Id-A1 and Id-B4 (both BALB/c IgG1), which are directed to the idiotypic determinants carried by the anti-Lewis Y mAb, AH-6 (BALB/c IgM). Both Id-A1 and Id-B4 (Ab2) recognized paratope-related idiotopes carried by the AH-6 antibody (Ab1); they specifically inhibited the binding of AH-6 to the Lewis Y Ag. The high idiotypic connectivity of anti-Lewis Y antibodies was noted; the polyclonal anti-idiotype antibody, produced in the sera of BALB/c mice by immunizing AH-6 antibody, cross-reacted with several anti-Lewis Y mAb which has been established in different laboratories. Id-B4 and Id-A1 seem to represent such cross-reactive anti-idiotypic antibodies. Id-A1 recognized an idiotope carried by two out of six panel Ab1 mAb directed to the Lewis Y Ag. Id-B4 reacted with four out of the six panel antibodies, and was considered to recognize a recurrent idiotope of anti-Lewis Y antibodies which occurs more commonly than the idiotope recognized by Id-A1. All of the anti-Lewis Y antibodies which carry idiotopes that react with Id-A1 or Id-B4 were encoded by the VH genes of the VH7183 family; the most D-J proximal VH gene family in BALB/c mice, which is known to be preferentially expressed in embryonic B cells. Immunization of BALB/c mice with keyhole limpet hemocyanin-conjugated Id-B4 and/or Id-A1 induced a significant titer of anti-Lewis Y antibodies (Ab1-like Ab3) in the sera.

Animals↗

Embryonic development of rat diaphragm. An electron-microscopic study.

Ultrastructural aspects of white Wistar rats diaphragm during part of its embryonic development (from the 13th embryonic day till birth) have been studied. The dominating structures observed in the period of the thirteenth embryonic day (ED 13) are undifferentiated cells, their cytoplasm being poor in organelles but rich in ribosomes. The close examination of these cells reveals that some of them possess a kind of thin filaments near their Golgi zones. At ED 14-15 clusters of myoblasts are readily detected (their cytoplasm containing a lot of glycogen granules and myofibrils, some of them even with Z-line material); contact sites between their cell membranes appear, somewhere forming specialized junctions; in this period the myoblasts start to fuse giving rise to the primary generation of myotubes. At ED 16-17 the quantity of myofilaments and glycogen granules increases alongside with the initiation of a basal-lamina formation; occasionally some oval, undifferentiated cells very similar to those viewed at ED 13 are found. At ED 18-19 the cytoplasm of the myotubes contains a lot of myofibrils and a well-developed endoplasmic reticulum; at some places the adjacent membranes still form deep interdigitations. At the end of the prenatal myogenesis (ED 20-21) most of the muscle cells are close to their mature morphological appearance--the sarcomers are well-organized and some nuclei present a peripheral localization; nevertheless, in this period new generations of myotubes can be also distinguished.

Animals↗

Three-dimensional ultrasonography in the first trimester of human pregnancy.

Our purpose was to visualize normal embryonal and fetal surface anatomical structures in the first trimester of human pregnancy by use of three-dimensional ultrasonography with a specially developed abdominal three-dimensional transducer. Four embryos and 31 fetuses of 8-13 weeks gestation were studied with a specially-developed abdominal three-dimensional transducer (3.5 MHz). This imaging system can provide conventional two-dimensional ultrasonography images and can also generate, within seconds, high-quality three-dimensional images in the surface and transparent mode with no need for an external workstation. The percentage of surface anatomical structures visualized at each gestational age interval using two-dimensional and three-dimensional ultrasonography is presented. Head and trunk were depicted in all cases. The number and the clarity of visualization of face, upper and lower extremities, hand, and foot increased with advancing gestation. The free loop of the umbilical cord was depicted in most cases. The number of depictions of abdominal cord insertion, midgut herniation, and yolk sac decreased with the increase of gestation. Genitals could not be identified in the first trimester. The ability to view some surface anatomical structures (face, hand, and foot) was better with three-dimensional ultrasonography than with two-dimensional ultrasonography. Three-dimensional ultrasonography provides a novel means for visualization of surface anatomical structures of the embryo and early fetus. These results suggest that three-dimensional ultrasonography can become an important modality in future embryological and early fetal research and in detection of embryonic and fetal developmental disorders in the first trimester of pregnancy.

Cross-Sectional Studies↗

In vitro polymerization of embryonic MAP-2c and fragments of the MAP-2 microtubule binding region into structures resembling paired helical filaments.

The microtubule-associated protein Tau is widely regarded as the principal component of paired helical filaments comprising Alzheimer neurofibrillary tangles. Tau fragments containing the non-identical repeat region formed structures resembling paired helical filaments (Schweers, O., Mandelkow, M., Biernat, J., and Mandelkow, E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8463-8467). MAP-2, the other structurally related neuronal microtubule-associated protein, has not been implicated in paired helical filament formation. We now describe the assembly of paired helical filament-like structures from MAP-2 polypeptides containing only 100 residues. A dimeric species, stabilized by an interchain disulfide, appears to be involved in the assembly reaction. We also investigated the polymerization of embryonic MAP-2c, which, except for its microtubule binding region, is structurally distinct from Tau. Full-length MAP-2c formed paired helical filament-like polymers. Polymerized MAP-2c and the microtubule binding region fragment readily bound thioflavin-S, a dye that stains paired helical filaments in the histochemical diagnosis of Alzheimer's disease. Our unprecedented finding that a small MAP-2 microtubule binding region fragment and MAP-2c can form structures resembling straight filaments or Pronase-treated paired helical filaments raises fundamental questions concerning the role of MAP-2 in the pathobiology of Alzheimer disease.

Amino Acid Sequence↗

X-ray microanalysis of developing and mature inner ear.

X-ray microanalysis technique is a new tool in inner ear research. According to the literature in this field only the energy dispersive type of analysis has been used. Data obtained from X-ray microanalysis are in agreement with results using other techniques, mainly fluid withdrawal from the endo- and perilymphatic spaces. The introduction of serial sectioning (16 microns thick sections) of the temporal bone for microanalysis now also permits analysis of embryonic material with its delicate structures too small for other techniques. The energy dispersive technique is thus suitable for analysis of both biological fluids and tissues. Studies have so far been focused on mainly normal conditions in the embryonic and adult labyrinth.

Aging↗

Placental ontogeny of the Tasmanian scincid lizard, Niveoscincus ocellatus (Reptilia: Squamata).

A prominent scenario for the evolution of reptilian placentation infers that placentotrophy arose by gradual modification of a simple vascular chorioallantoic placenta to a complex structure with a specialized region for nutrient transfer. The structure of the chorioallantoic placenta of Niveoscincus ocellatus, apparently described originally from a single embryonic stage, was interpreted as a transitional evolutionary type that provided support for the model. Recently, N. ocellatus has been found to be as placentotrophic as species with complex chorioallantoic placentae containing a specialized region called a placentome. We studied placental development in N. ocellatus and confirmed that the chorioallantoic placenta lacks specializations found in species with a placentome. We also found that this species has a specialized omphaloplacenta. The chorioallantoic placenta is confined to the region adjacent to the embryo by a membrane, similar to that found in some other viviparous skinks, that divides the egg into embryonic and abembryonic hemispheres. We term this structure the "inter-omphalopleuric" membrane. The position of this membrane in N. ocellatus is closer to the embryonic pole of the egg than to the abembryonic pole and thus the surface area of the omphaloplacenta is greater than that of the chorioallantoic placenta. In addition, the omphaloplacenta is regionally diversified and more complex histologically than the chorioallantoic placenta. An impressive and unusual feature of the omphaloplacenta of N. ocellatus is the development of extensive overlapping folds in the embryonic component of mid-gestation embryos. The histological complexity and extensive folding of the omphaloplacenta make this a likely site of placental transfer of nutrients in this species.

Allantois↗

[Self-organization in the determination of the size of the axial structures in the embryogenesis of the clawed toad].

Experiments were performed using X. laevis embryos during gastrulation and neurulation (stages 10, 11 1/2, 12 1/2, 13 1/2, 15 and 18). Part of presumptive epidermis and lateral plate mesoderm was removed, and embryos raised until stage 25. The size of axial structures (notochord, somite mesoderm, central nervous system) was determined using serial histological sections and compared with that of control embryos. In experimental embryos, the size of axial structures was decreased. Until a specific stage of development, close correlation was found between the volume of embryonic compartment corresponding to a particular, structure and the volume of presumptive epidermis and lateral plate mesoderm. This stage is individual for each axial organ: middle gastrula (stage 11 1/2) for notochord, late gastrula (stage 12 1/2) for somite mesoderm, and late neurula (stage 18) for central nervous system. This data suggest that differentiation pattern of ecto-mesodermal rudiment is subject to regulation during gastrulation-neurulation, and subdivision of ectoderm and mesoderm into axial and non-axial tissues is a self-organizing process.

Animals↗

Unusual structural features and assembly of gap and pleated septate junctions in embryonic cockroach CNS.

Junctional assembly in the developing CNS in cockroach embryos has been studied during the last half of neurogenesis. Atypical linear tracts of gap junctions are found to develop between attenuated cytoplasmic glial cell processes and their overlying perineurial cells during the last third of development. During both perineurial and glial gap-junctional formation, 13 nm E face (EF) intramembrane particles (IMPs), such as are characteristic of arthropod gap junctions, are seen initially as free IMPs; these then become arranged in loose irregular clusters or alignments and finally are aggregated in plaques. P face ridges (or EF grooves), typical of tight junctions, are found on the same perineurial membrane face as assembling gap-junctional PF pits (or EF particles). Pleated separate junctions also develop between adjacent perineurial processes during the last third of embryogenesis; these form by the apparent migration of individual 8 nm PF IMPs into meandering rows, which then become aligned in numerous orderly parallel stacks. Although all these junctions occur on the same perineurial membrane face, the IMPs that form the different junctional types never appear to be confused during junctional assembly. The cues to signal the advent of these precise patterns, however, are unknown.

Animals↗

[The structural characteristics of the dendritic apparatus of transplanted neurons from the embryonic amygdala].

Two main neuronal classes were studied in the grafts of basolateral nucleus of the rat embryonic amygdala stained by the Golgi method--sparsely and densely ramified cells. Transplantation resulted in the increase of dendrite length and ramification of sparsely ramified cells, in the decrease of cell body size and increase of ramification of densely ramified cells. The analysis of polar histograms of the dendritic orientation shows the selective increase of dendrite length and ramification of the both neuronal classes. The dendrites of the intact neurons of basolateral amygdala are distributed almost regularly, they do not display any dominant orientation. After transplantation the main orientation to the area of graft-host integration appears. The changes in dendrites are discussed with respect to the ability of the graft to take part in compensation of the damaged brain functions.

Amygdala↗

Retrovirus like particles produced by human embryonal cells and cell lines derived from human malignancies. II. Protein structure.

Very small amounts of retrovirus-like particles were isolated from tissue culture media of various types of human cell lines derived from malignant as well as normal cells. The aim of the present study is the characterization of protein profiles of these isolates. The comparison of protein profiles of isolated human virus-like particles with the profiles of well characterized animal as well as human exogenous (LAV/HTLV-III) retroviruses revealed a 25k protein (p25) to be a major protein or at least one of the protein components of human retrovirus-like particles.

Cell Line↗

Structural components and characteristics of Reichert's membrane, an extra-embryonic basement membrane.

This paper presents an analysis of mouse and rat Reichert's membrane, a thick basement membrane formed between the trophoblast and parietal endoderm cells of early mammalian embryos. When analyzed by polyacrylamide gel electrophoresis. Reichert's membranes from rat and mouse conceptuses appear simple and consist primarily of collagen and 4 noncollagenous glycoproteins of approximate Mr = 415,000, 245,000, 170,000, and 50,000. The proteins at 415,000 and 245,000 are similar in molecular weight to laminin and are immunoprecipitated by anti-laminin antiserum. The protein at 170,000 is co-precipitated with laminin and represents a novel form of a laminin-like protein, whereas the protein at 50,000 is unrelated to laminin. Using metabolic labeling experiments, it is shown that parietal endoderm cells synthesize these components and incorporate them into the matrix in organ cultures but do not degrade Reichert's membrane in the conditions used over a 6-day culture period. Evidence is presented that the high molecular weight collagenase-sensitive proteins (greater than 420,000) observed on polyacrylamide gel electrophoresis are due to lysyl oxidase-derived cross-links between Reichert's membrane components.

Animals↗

F-spondin and mindin: two structurally and functionally related genes expressed in the hippocampus that promote outgrowth of embryonic hippocampal neurons.

Extracellular matrix (ECM) proteins play an important role in early cortical development, specifically in the formation of neural connections and in controlling the cyto-architecture of the central nervous system. F-spondin and Mindin are a family of matrix-attached adhesion molecules that share structural similarities and overlapping domains of expression. Genes for both proteins contain a thrombospondin type I repeat(s) at the C terminus and an FS1-FS2 (spondin) domain. Both the vertebrate F-spondin and the zebrafish mindins are expressed on the embryonic floor plate. In the current study we have cloned the rat homologue of mindin and studied its expression and activity together with F-spondin in the developing rodent brain. The two genes are abundantly expressed in the developing hippocampus. In vitro studies indicate that both F-spondin and Mindin promote adhesion and outgrowth of hippocampal embryonic neurons. We have also demonstrated that the two proteins bind to a putative receptor(s) expressed on both hippocampal and sensory neurons.

Amino Acid Sequence↗

The 5' terminal capping of heterogeneous nuclear RNA at different embryonic stages of the sea urchin.

5' Terminal cap structures of hnRNA have been characterized and the extent of capping determined as a function of embryonic development. Sea urchin embryo hnRNA contains only the type-1 cap, m7GpppNmpNp, with the type-2 cap, which has a 2'-0-methylated subpenultimate nucleotide, being associated only with stable small nuclear RNAs. These cap 2-containing RNAs are synthesized at a rate of approximately 70 molecules min-1 nucleus-1 compared to approximately 1000 molecules for hnRNA cap 1. Approximately 70% of nuclear cap 1 is associated with greater than 15S RNA in denaturing solvent, but under non-denaturing conditions the percentage is much higher. Cap 1 in low and high molecular weight nuclear RNA have the same kinetics of methyl labeling. Thus all cap 1 structures may belong to a single class either covalent or H-bonded to high molecular weight RNA. hnRNA greater than 15S is 35% capped; however, adding caps in less than 15S RNA gives an estimate of 50% capping for total hnRNA. In development from early blastula to late gastrula, there is little if any change in the extent of capping of hnRNA. These results coupled with others indicate that the fraction of hnRNA molecules serving as precursor to mRNA does not change quantitatively during embryonic development.

Animals↗