Quantitative Dissection of the Proximal Enhancer.

A major goal in biology is to understand the rules by which cis-regulatory sequences control spatially and temporally precise expression patterns. Here we present a systematic dissection of the proximal enhancer for the notochord-specific transcription factor in the ascidian chordate . The study uses a quantitative image-based reporter assay that incorporates a dual-reporter strategy to control for variable electroporation efficiency.

Evolution of Developmental Programs for the Midline Structures in Chordates: Insights From Gene Regulation in the Floor Plate and Hypochord Homologues of Embryos.

In vertebrate embryos, dorsal midline tissues, including the notochord, the prechordal plate, and the floor plate, play important roles in patterning of the central nervous system, somites, and endodermal tissues by producing extracellular signaling molecules, such as Sonic hedgehog (Shh). In , , one of the two genes, is expressed in the floor plate of the embryonic neural tube, while none of the genes are expressed in the notochord. We have identified a -regulatory region of that was sufficient to drive a reporter gene expression in the floor plate.

Brachyury controls notochord fate as part of a feed-forward network.

The notochord is a defining feature of the chordates. The transcription factor Brachyury (Bra) is a key regulator of notochord fate but here we show that it is not a unitary master regulator in the model chordate Ectopic Bra expression only partially reprograms other cell types to a notochord-like transcriptional profile and a subset of notochord-enriched genes is unaffected by CRISPR Bra disruption. We identify Foxa.

Cellular identity and Ca signaling activity of the non-reproductive GnRH system in the Ciona intestinalis type A (Ciona robusta) larva.

Tunicate larvae have a non-reproductive gonadotropin-releasing hormone (GnRH) system with multiple ligands and receptor heterodimerization enabling complex regulation. In Ciona intestinalis type A larvae, one of the gnrh genes, gnrh2, is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord, respectively, of vertebrates. The gnrh2 gene is also expressed in the proto-placodal sensory neurons, which are the proposed homologue of vertebrate olfactory neurons.

Heterotopic pregnancy with suspicion of superfetation after the intrauterine insemination cycle with ovulation induction using clomiphene citrate: A case report.

At 22 days after intrauterine insemination with ovulation induction using clomiphene citrate at a previous hospital, a 30-year-old woman was admitted to our hospital owing to right lower quadrant abdominal pain. We diagnosed threatened abortion because of a gestational sac in the uterus on transvaginal ultrasonography. The next day, she complained of increased abdominal pain.

The Use of cis-Regulatory DNAs as Molecular Tools.

Ascidians possess relatively small and compact genomes. This feature enables us to easily isolate cis-regulatory DNAs of genes of interest. Particularly, cis-regulatory DNAs of genes showing tissue- or cell-type-specific expression are routinely used for the artificial induction of gene expression.

Constrained vertebrate evolution by pleiotropic genes.

Despite morphological diversification of chordates over 550 million years of evolution, their shared basic anatomical pattern (or 'bodyplan') remains conserved by unknown mechanisms. The developmental hourglass model attributes this to phylum-wide conserved, constrained organogenesis stages that pattern the bodyplan (the phylotype hypothesis); however, there has been no quantitative testing of this idea with a phylum-wide comparison of species. Here, based on data from early-to-late embryonic transcriptomes collected from eight chordates, we suggest that the phylotype hypothesis would be better applied to vertebrates than chordates.

Revised lineage of larval photoreceptor cells in Ciona reveals archetypal collaboration between neural tube and neural crest in sensory organ formation.

The Ciona intestinalis larva has two distinct photoreceptor organs, a conventional pigmented ocellus and a nonpigmented ocellus, that are asymmetrically situated in the brain. The ciliary photoreceptor cells of these ocelli resemble visual cells of the vertebrate retina. Precise elucidation of the lineage of the photoreceptor cells will be key to understanding the developmental mechanisms of these cells as well as the evolutionary relationships between the photoreceptor organs of ascidians and vertebrates.

The Inequality of Patient Profile Information in Japanese Hospitals.

A model dataset of patient profile information was created based on the items used at five Japanese university hospitals, the patient information data elements in Health Level 7 (HL7) v2.5, and the standard datasets for medical information exchange used in Japan. In order to check the validity of the model dataset, a cross-sectional survey was performed.

Genome-wide identification and characterization of transcription start sites and promoters in the tunicate Ciona intestinalis.

The tunicate Ciona intestinalis, an invertebrate chordate, has recently emerged as a powerful model organism for gene regulation analysis. However, few studies have been conducted to identify and characterize its transcription start sites (TSSs) and promoters at the genome-wide level. Here, using TSS-seq, we identified TSSs at the genome-wide scale and characterized promoters in C.

Upstream regulatory sequences required for specific gene expression in the ascidian neural tube.

The relatively simple structure of ascidians and the number of associated molecular resources that are available make ascidians an excellent experimental system for Investigating the molecular mechanisms underlying neural tube formation. The ascidian neural tube demonstrates the same basic morphology as that of vertebrates. We have described the expression of the neural tubespecific gene CiNut1, which is expressed within neural tube precursor cells from the gastrula stage, and along the entire length of the neural tube during its formation.

Disruption of neuronal migration in the neocortex of the dreher mutant mouse.

To analyze developmental abnormalities related to neuronal migration in the dreher mutant mouse, the neocortical cytoarchitecture of dreher and control mice were examined in Nissl-stained serial sections by light microscopy. In general, the dreher neocortex has six layers which are similar in size and thickness to those observed in normal mouse neocortex. However, in dreher neocortex, three types of abnormalities were found: (1) an increase in the number of diffusely distributed neurons in layer I, (2) small, ectopic collections of neurons in layer I, and (3) isolated disturbances of local cytoarchitecture characterized by neuron-free space distributed in areas between layer II to IV.

Abnormal distribution of acetylcholinesterase activity in the hippocampal formation of the dreher mutant mouse.

The distribution of acetylcholinesterase(AChE) in the hippocampal formation of the dreher mutant mouse was studied by comparing homozygous mutant (drsst-J/drsst-J) with littermate control (+/? or +/+). In the control mice, AChE activity was most intense in the inner one-third of the stratum oriens and lacnosum of the hippocampus, and in the inner one-fifth of the molecular layer of the dentate gyrus. In contrast, in homozygous dreher mice, AChE activity in area CA3c of the hippocampus was not restricted to the stratum oriens, and extended upward into the infrapyramidal and suprapyramidal mossy fiber layers, the lower part of the stratum radiatum, the pyramidal cell layer, and downward toward the alveus.

Prominent expression of type 1 (gamma) protein kinase C in Purkinje cells located in the "central mass" of Reeler mutant mouse cerebellum as revealed by a computer image analysis technique.

In Reeler mutant mice, cerebellar Purkinje cells exhibit abnormal synaptogenesis. In this study, Purkinje cells in the "central mass" with abnormal afferent connections were compared with those located in their normal position in the cortex in terms of immunoreactivity for type 1 (gamma) protein kinase C. A "computer image analysis technique" was developed for the purpose of this quantitative study, and it revealed that the immunoreactivity in the central mass was significantly higher than that in the cortex.

Cytoarchitectonic abnormalities in hippocampal formation and cerebellum of dreher mutant mouse.

The laminated structures in the hippocampal formation and cerebellum of homozygous dreher mice were compared to their littermates and to C57BL/6J mice in Nissl- and myelin-stained preparations. In the dreher dentate gyrus, ectopic granule cells were situated in the molecular layer, and frequently there was either partial or complete absence of the infrapyramidal limb of the granule cell layer. In the dreher hippocampus, the cells of the pyramidal cell layer in area CA3 formed widely dispersed arrangements, and there were ectopically situated pyramidal cells in the stratum radiatum and stratum oriens.

Cytoarchitectonic abnormalities in cerebellum and hippocampal formation of beige mutant mouse.

Abnormalities in the cerebellum and hippocampal formation of the beige mouse were revealed by histological and immunohistochemical examination. In the cerebellum, Purkinje cells and clusters of granule cells, plus an occasional Bergmann glia cell, were located ectopically in the molecular layer. In the hippocampus, ectopically situated pyramidal cell were found in the stratum oriens of area CA3.

Abnormalities of foliation and neuronal position in the cerebellum of NZB/BINJ mouse.

To analyze developmental abnormalities related to neural migration in the NZB/BINJ mouse, the pattern of cerebellar foliation and neural position were compared with that of a normal mouse (C57BL/6J). Three abnormalities of cerebellar foliation--(1) lobe isolated from other cerebellar lobes, (2) lobes imbalanced in relative amounts or ratio of granular cell layer and molecular layer, (3) lobes in which some Purkinje cells and the molecular layer was embedded in the granular cell layer--were observed in NZB/BINJ mice. These morphological abnormalities were not limited to a specific lobe.

Histochemical demonstration of heavy metals in the hippocampal formation embedded in Quetol 523M.

To facilitate improvement of investigations on the distribution of mossy fibers in the hippocampal formation, a method is described using Timm's stained preparations after methacrylate embedding with the hydrophilic resin, Quetol 523M. Fixation with a mixture of formaldehyde and glutaraldehyde yielded satisfactory staining results and good structural preservation. During the course of histochemical experiments employing Timm's staining, examinations revealed that sulfide silver reaction products were consistently present in both the mossy fibers themselves and their terminals associated with the dendrites of pyramidal cells in tissue sections of 1-2 microns in thickness.

Correlative light and electron microscopic observations on ectopic neurons in the cerebellum of dreher mutant mouse.

The structure of ectopic neurons in the cerebellum of dreher mutant mouse was investigated by correlative light and electron microscopic observations. Tissue blocks were fixed in buffered aldehyde and embedded in a mixture of 2-hydroxypropyl methacrylate, Quetol 523, and methyl methacrylate. Sections at 0.

Staining of intestinal goblet cells with ruthenium red in semithin sections.

For a correlative light and electron microscopy of intestinal goblet cells, postembedding staining with ruthenium red (RR) was performed in epoxy-embedded sections. Tissue blocks were fixed in buffered aldehyde and embedded in a mixture of Quetol 651, nonenyl succinic anhydride (NSA), methyl nadic anhydride (MNA), and DMP-30. Sections at 0.

Use of semithin sections embedded in a water-miscible methacrylate for light microscopy of central nerve tissues.

Semithin sections embedded in water-miscible methacrylates were used for the study of fine structures of cells and tissues in the central nervous system by light microscopy instead of the conventional paraffin sections. This method used a water-miscible methacrylate mixture consisting of 2-hydroxypropyl methacrylate (HPMA), Quetol 523 and methyl methacrylate (MMA) as an embedding medium. The mixture had a low viscosity, was easy to handle and penetrated readily and completely into the specimen, producing a homogenous block from which it was easy to make sections 1.

Quantitative analysis of synaptic boutons on motoneurons of rhesus monkey spinal cord after chronic deafferentation due to cerebral lesions.

The motoneurons in the lateral nucleus of the anterior horn of the rhesus monkey cervical enlargement were reconstructed by light microscopy of semi-thin sections of 2 micro in thickness which were cut serially, alternately with ultra-thin sections for electron microscopy. Four single neurons were reconstructed with a computer graphic system, and simultaneously the synaptic boutons on the soma and each dendrite of single reconstructed neurons were analyzed quantitatively by electron microscopy. The synaptic boutons were classified into 4 types on the basis of the shape of the synaptic vesicles.