Spatiotemporal regulation of fibroblast growth factor signal blocking for endoderm formation in Xenopus laevis

We have previously shown that the inhibition of fibroblast growth factor (FGF) signaling induced endodermal gene expression in the animal cap and caused the expansion of the endodermal mass in Xenopus embryos. However, we still do not know whether or not the alteration of FGF signaling controls embryonic cell fate, or when FGF signal blocking is required for endoderm formation in Xenopus. Here, we show that FGF signal blocking in embryonic cells causes their descendants to move into the endodermal region and to express endodermal genes. It is also interesting that blocking FGF signaling between fertilization and embryonic stage 10.5 promotes endoderm formation, but persistent FGF signaling blocking after stage 10.5 restricts endoderm formation and differentiation.

Screening of Interacting Proteins with PV.1 as Downstream Factors of BMP Signal / 대한해부학회지

Homeodomain transcription factors functioning downstream of BMP ventral pathway have been reported to share similar domain of roles in mesoderm patterning along the dorsal-ventral axis. To elucidate the differential role of PV.1 in the aspect of relationship between dorsal and ventral region, we tried to screen PV.1- interacting proteins. Twenty-four PV.1-interacting proteins were identified by yeast two-hybrid screening. Xvent-2 and Xclaudin-6 among these, went under domain study. The C-terminus of PV.1, more specifically 197-241 region was found to interact with Xclaudin-6. Meanwhile Xvent-2 has mild affinity to overall C-terminal region of PV.1. At the same time it was found that Xvent-2 homodimerizes and also binds to Xclaudin-6.

Transcriptional regulation of Zic3 by heterodimeric AP-1(c-Jun/c-Fos) during Xenopus development

The heterodimeric c-Jun/c-Fos, an activator protein-1 (AP-1) has been implicated in mesoderm induction (Dong et al., 1996; Kim et al., 1998) whereas the homodimer of c-Jun was reported to be involved in neural inhibition during the early development of Xenopus embryos. During the early vertebrate development AP-1 involvement in the neural induction is still not clearly understood. We report here that AP-1 has a role in Zic3 expression, a critical proneural gene and a primary regulator of neural and neural crest development (Nakata et al., 1997; Nakata et al., 1998). AP-1 was able to induce the Zic3 gene in a dose dependent manner but other homo- or hetero-dimeric proteins, such as c-Jun/c-Jun, JunD/FosB or JunD/Fra-1 were not. The inhibition of AP-1 activity using morpholino antisenses of c-jun mRNAs blocked the Zic3 expression induced by activin. In addition, co-injection of c-jun mRNA rescued the down-regulated Zic3 expression. The promoter region of isolated Zic3 genomic DNA was found to possess several consensus-binding site of AP-1. Thus, in the functional assays, AP-1 could increase promoter activity of Zic3 gene. These findings suggest that proneural gene, Zic3 may be regulated by heterodimeric AP-1(c-Jun/c-Fos) and it may have a role in activin signaling for the regulation of neural specific gene, Zic3.

Quantitative Ultrastructural Analysis of Periodontal Afferent Terminals in the Trigeminal Motor Nucleus / 대한해부학회지

Little is known about processing mechanism of sensory input from the periodontal ligaments to the trigeminal motor nucleus for the control of chewing force and modulation of chewing pattern. Low threshold mechanoreceptive periodontal afferent was labeled with horseradish peroxidase by use of intra-axonal injection technique and investigated with electron microscopy. Quantitative ultrastructural analysis was performed on the 39 serially reconstructed labeled boutons in the trigeminal motor nucleus in cat. Labeled bouton contained clear spherical vesicles and one or two large dense cored vesicles. Most of labeled boutons were dome or round shape. All the analysed labeled boutons were presynaptic to dendritic shaft or distal dendrite and those presynaptic to soma or proximal dendrite were not observed. A large number of labeled boutons (46.2%) were postsynaptic to one or two presynaptic pleomorphic vesicle containing endings. Synaptic triad, in that a presynaptic ending which is presynaptic to the labeled bouton, in turn, is presynaptic to dendrite that is postsynaptic to the labeled bouton, was observed in 10.3% of the labeled boutons. Most of the labeled boutons showed simple synaptic organization, in that 64.1% of the labeled boutons made synaptic contacts with one or two neuronal profiles. One (2.6%) of the 39 analyzed labeled boutons showed synaptic contacts with 5 or more neuronal profiles. Labeled bouton volume, mitochondrial volume, apposed surface area and active zone area showed wide variation. These ultrastructural parameters were positively correlated with bouton volume. The values for apposed surface area and active zone area with presynaptic p-endings, in contrast to those with postsynaptic dendrites, showed narrow range and had little correlation with bouton volume. The present study revealed characteristic features on ultrastructural parameters of labeled boutons from periodontal afferent which is involved in periodontal masseteric reflex, and that influence on the postsynaptic trigeminal motoneurons showed wide variability.

Temporal Inhibition of FGF Signal on Endoderm Formation during Early Xenopus laevis Embryogenesis / 대한해부학회지

Our previous results showed that FGF signaling, which is important for the mesoderm and neuroectoderm induction, should be blocked for the endoderm formation in Xenopus. Here, Xenopus embryos were collected according to the two time points of MBT or stage 10.5. FGF signal was blocked with SU5402, chemical inhibitor of FGF signal, in the stage-specific embryos, to understand the role of FGF signal during the endoderm formation in the stage-specific embryos. Embryos subjected with the blocking of FGF signal before stage 10.5 showed the expanded abdominal volume in which endodermal mass was increased about 2 times but abdominal organs were not found. The tissue recombinant experiment showed that mesodermal tissue was necessary for the differentiation of endoderm. Embryos subjected with the blocking of FGF signal after stage 10.5 showed that abdomen was not expanded, the neural tube was opened instead. Our data indicate that blocking of FGF signal before stage 10.5 may be necessary for the endoderm induction and signals from neighboring endoderm tissue and mesoderm are required for the endoderm differentiation.

Processing Mechanism of Sensory Information Originated from the Oral Cavity in the Trigeminal Nucleus Oralis / 대한해부학회지

To analyze the synaptic characteristics of axon terminals originated from the tooth pulp in the trigeminal nucleus oralis, labeling of tooth pulp afferents with wheat-germ agglutinin conjugated horseradish peroxidase and morphometric analysis with electron microscopic photographs were performed. The results obtained from 23 labeled endings were as follows. All of the labeled boutons contained clear and round synaptic vesicles (dia. 45~55 nm). 3 (13.64%) out of 23 labeled endings have 20~105 dense cored vesicles and do not make synaptic contacts with p-endings. But remaining 20 labeled endings (86.36%) almost do not have dense cored vesicles and 12 of them make synaptic contacts with p-endings. The mean number of synaptic contacts was 2.61+/-2.06 and the postsynaptic profiles were usually middle or distal dendrite and dendritic spine (1.74+/-1.36) rather than soma or proximal dendrite. The mean number of synaptic contacts with pendings was 0.87+/-1.01. And the frequency of the synaptic triads were 0.39+/-0.58. The vesicle density was 993.23+/-267.41/mum(2). The volume of labeled bouton was 3.54+/-2.20 mum(3) and highly correlated (P < 0.01) with surface area (11.78+/-4.92 mum(2), r = 0.95), total apposed surface area (2.90+/-1.56 mum(2), r=0.72), total active zone area (0.61+/-0.37 mum(2), r = 0.82), mitochondrial volume (0.75+/-0.53 mum(3), r = 0.94), the number of synaptic vesicles (2621.30+/-1473.61, r= 0.91) and the number of synaptic contacts (r = 0.76). These results suggest that there are two groups of tooth pulp afferent terminals according to the presence of dense cored vesicles in the trigeminal nucleus oralis. And the sensory processing mechanism of each groups may be different. And the "size principle" of Pierce & Mendell (1993) is also applicable to these terminals.

Colocalization of ANG II and mRNA for the Renin-Angiotensin System Components in Cultured Rat Glomerular Epithelial and Mesangial Cells / 대한해부학회지

Mesangial cells are found to have renin and angiotensin II-AT1 receptors, but the presence of other components of the renin-angiotensin system and production of angiotensin II within the cell have not been demonstrated. The presence of the renin-angiotensin system components in the glomerular epithelial cell has not been previously reported. We studied expression of each component of the renin-angiotensin system in primary cultured rat glomerular epithelial cells and mesangial cells. We assessed mRNA expression by RT-PCR and the presence of angiotensin II by immunocytochemistry. Both cultured glomerular epithelial cells and mesangian cells expressed mRNA for components of the renin-angiotensin system such as renin, angiotensinogen and angiotensin II type 1A and 1B receptor subtypes. Immunocytochemical studies with specific antibody for angiotensin II demonstrated significant immunoreactivity in both glomerular epithelial cells and mesangian cells. These results, for the first time, provide direct evidence that both the glomerular epithelial cells and mesangian cells contain a complete renin-angiotensin system and generate angiotensin II with intracellular mechanisms. Further studies are required to define the subcellular localization of angiotensin II with electron microscopy and to elucidate the physiological importance of the intracellular reninangiotensin system.

Transcriptional Regulation of the Xbr-1a/Xvent-2 Gene by BMP-4 Signaling during Xenopus Embryonic Development / 대한해부학회지

BMP-4 signaling is mediated through Smad proteins which may translocate to the nucleus to activate transcription. Little is known about how BMP-4 signaling regulates the transcription of its target genes, e.g., Xvent genes. Therefore, we isolated the genomic clone of a BMP-4 responsive homeobox gene, Xbr-1a/Xvent-2. This clone contains a promoter and three exons for the entire coding region. Using the primer extension, we identified the transcription initiation site corresponding to position -64 bp upstream to the ATG codon of the Xvent-2 gene. The promoter was linked to the luciferase reporter gene, and promoter activity determined by luciferase assay. The temporal promoter activity peaked between embryonic stages 13~17, in agreement with its temporal mRNA expression in the whole embryo. Through the serial deletion mutation, the upstream -235 bp of the promoter retains the full transcriptional activity, and is regulated by BMP-4 signaling. The present results suggest that the BMP-4 responsive element is located on the upstream 235 bp of the promoter.

CGRP Immunoreactivities Following Artificail Ureteral Calsulosis and Ureteral Chemical Irritation in the Rat / 대한해부학회지

The aim of the present study was to investigate the CGRP-IR pattern in the ureter in stone-implanted rats and ureteral formalin irritated rats. Artificial ureteral stone rats were made according to Giamberardino's guide lines. For urethral formalin irritated rats, a small amount of diluted formalin was instilled into the ureter. The behavioral characteristics were observed and recorded with CCTV and analyzed, statistically. The rats showing characteristic visceral episode were sacrificed three days after stone implantation operation and after overnight chemical irritation followed by immunostaining of the ureter with anti-CGRP on the ureter In the artificial ureteral stone rats, CGRP IR fiber pattern showed variable changes on the upper portion of the stone implanted ureter. i.e., complete depletion, reticular pattern, and reticular pattern with more increased CGRP immunoreactivity were observed. In contrast, more-distinct CGRP IR fibers formed a reticular pattern but were not increased in density in the formalin irritated ureter. The results show the variable changing pattern of CGRP immunoreactivity in the ureter after artificial urethral calculosis, and the constant CGRP immunoreactivity in the ureter after chemical ureteral irritation.

Ultrastructural analysis of glutamate immunoreactive primary afferent terminals in the trigeminal nucleus principalis and trigeminal nucleus oralis of the rat / 대한해부학회지

The present study was aimed to investigate the ultrastructure of the primary afferent terminals and whether glutamate may be a transmitter in these terminals within the trigeminal nucleus principalis and oralis of the rat. Labeling of primary afferent terminals was performed by the injection of the CTB-HRP into the trigeminal ganglion. Ultrastructural analysis and assessment of the glutamate like immunoreactivity by the immunogold technique was performed with the 66 peroxidased labeled boutons in the nucleus principalis and 62 in the nucleus oralis. Labeled boutons were presynaptic to dendritic shafts of the secondary neurons and postsynaptic to the pleomorphic vesicles containing endings (p-endings). Most of the labeled boutons made synaptic contact with the dendritic shafts. A little labeled boutons in the nucleus oralis but no in the nucleus principalis was observed to make synaptic contact with the soma or proximal dendrite. Most of the labeled boutons made synaptic contact with one to three neurofiles, but labeled boutons showing complex synaptic connections, such as those with five or more neurofiles, were more in principalis than in oralis. The average diameter of p-endings were smaller than that of labeled boutons (p<0.05). The diameter of the postsynaptic dendritic shafts were smaller in nucleus principalis than in nucleus oralis, thus indicated that the labeled boutons made synaptic contact with more distal portion of the postsynaptic dendrite in the nucleus principalis than in the nucleus oralis. The gold particle density over the labeled boutons were significantly higher than that over the p-endings and average tissue particle density. They were ranged from 110 to 430% of the average tissue particle density. These findings indicate that synaptic connection of the primary afferent terminals is organized in different manner in nucleus principalis and oralis, and suggest that glutamate is involved as neuroactive substance in the primary afferent terminals of the trigeminal system.

Bucco-lingual Implant Path in the Posterior Mandible / 체질인류학회지

The purpose of this study is to investigate the cross -sectional anatomy of posterior mandibular body for proper determination of bucco -lingual implant path. Using fifty -four human mandibles, negative images of each mandible were made of agar impression material. The agar blocks were cut through the imaginary long axis of each root of three molars (M1M, M1D, M2M, M2D and M3). The depth of submandibular fossa, the angulation of long axis of mandibular body and tooth, and the length, angulation and ratio of alveolar bone superior to mylohyoid ridge and basal bone inferior to mylohyoid ridge were measured. The results obtained were as follows; 1. All of the measured angulations were decreased as it moves from M1M to M3. 2. The correlation coefficients among the angulation of the mandibular bone and the crown axis showed the high relationship (r=0.793), and the crown axis was steeper than the mandibular bone axis by 6.2 at M1M and 7.6 at M2M. 3. The length of upper alveolar bone was decreased, but that of inferior basal bone was increased as it moves from M1M to M3. 4. The depth of submandibular fossa was increased as it moves from M1M to M3. These results indicate that the angulation of implant path at the posterior mandible must be tilted more than wax -up crown axis by 6.2 at mesial root of 1st molar and by 7.6 at mesial root 2nd molar area for prevention of lingual cortical bone perforation during implant surgery.

Distribution of Tyrosine Hydroxylse Immunoreactive Structure in the Spinal Cord and Dorsal Root Ganglion of the Rat / 대한해부학회지

With the aim of gaining more insight into the catecholaminergic system in the nervous system of the rat, we have studied the precise distribution pattern of the tyrosine hydroxylase immunoreactive[TH-IR] fibers and soma in the spinal cord and dorsal root ganglion. In the dorsal root ganglion[DRG], TH-IR fibers were observed to run along the vessel wall, spirally and not found in the neural tissue itself. A few TH-IR fibers were found in the spinal nerve, not in the ventral root. Many TH-IR neurons were distributed in the L3, 4, 5, and 6 DRG but none of them were found in the other DRG segments. In the spinal cord, TH-IR fibers have shown sparse distribution all over spinal cord but relatively dense distribution in the ventral horn, intermediolateral column, lamina I of the dorsal horn of the cervical, lumbar, sacral, and coccygeal segment. TH-IR neurons were found in the intermediolateral column, dorsal gray commissure, dorsal horn of the C1 and C2 segments and S1-4 segments. TH-IR neurons in the cervical segments were polygonal and spindle shaped with well developed processes. In contrast to this, TH-IR neurons in the sacral segments were oval or spindle shaped with no processes. In conclusion, neurons in the DRG were not influenced by catecholaminergic nervous input. Intrinsic catecholaminergic nervous systems were found in both of spinal cord and DRG.

Distribution of Neuropeptide mRNA-Containing Neurons and Changes of Their Gene Expression in the Rat Periaqueductal Gray in a Neuropathic Pain Model / 대한해부학회지

The distribution of enkephalin, dynorphin, substance P and neurotensin in the periaqueductal gray[PAG] has been well established by immunohistochemical methods. However, there is little information about the regional distribution of these neuropeptide mRNA-containing neurons in the PAG. The present study was undertaken [1] to elucidate the distribution of these neuropeptide mRNA-containing neurons and to determine of the PAG, [2] to know how peptide expression relates to the proposed functional subdivisions of the PAG and [3] to know how neuropeptide mRNA levels in the PAG change following peripheral neuropathy The results obtained are as follows ; 1. Preproenkephalin[pENK] mRNA-containing neurons are found mostly in the ventrolateral portion at all levels of the PAG. 2. Prodynorphin[pDYN] mRNA-containing neurons are concentrated mostly in the ventrolateral portion at the caudal level of the PAG. 3. Preprotachykinin[pTAK] mRNA-containing neurons are localized mainly in the ventrolateral portion at all levels of the PAG. There is small numbers of pTAK mRNA-containing neurons in the dorsolateral and dorsal portion at all levels of the PAG. 4. Proneurotensin[pNT] mRNA-containing neurons are concentrated mostly in the medial part of ventrolateral portion of the caudal and mid PAG. 5. Peripheral neuropathy induces an increase of pNT mRNA levels in the PAG, while pENK, pDYN and pTAK mRNAs levels show no change. The present results indicate that the pENK, pDYN, pTAK or pNT mRNA-containing neurons are found mainly in the ventrolateral PAG, the area where analgesia is most easily produced and that neurotensin in the PAG may play an important role in modulating chronic neuropathic pain.

CNS innervation of the urinary bladder demonstrated by immunohistochemical study for c-fos and pseudorabies virus

The aim of the present study is to verify the functional and anatomical neural pathways which innervate the urinary bladder in the central nervous system of the rat. To identify the functional neural pathway, the urinary bladder was stimulated by infusing formalin for 2 h. Then, brain and spinal cord were dissected out and immunohistochemistry was done by using anti-c-fos antibody. Many c-fos immunoreactive (IR) neurons were identified in the telencephalic cortical areas and in several brainstem nuclei, which are known mostly to be related with urinary bladder. In the spinal cord, a number of c-fos IR neurons were found in the lamina I, IIo, dorsal gray commissure, sacral parasympathetic nucleus. To identify the anatomical neural pathway of the urinary bladder, Pseudorabies virus (PRV) was injected into the wall of urinary bladder and was identified with anti-PRV by using immunohistochemistry. Most PRV labeled neurons were found where c-fos IR neurons were identified and few of them were also in the areas where c-fos IR neurons were not found, e.g., prefrontal cortex, agranular insular cortex, and subfornical organ. In the spinal cord, PRV labeled cells were found all over the gray matter. The present study presents morphological evidence demonstrating the supraspinal areas are related with the neural control of the urinary bladder and most functional neural pathway of the urinary bladder is well consistent with the anatomical neural pathway except in some telencephalic cortical areas.

Effect of loss of incisal function on the growth activities and ultrastructure of the condylar cartilage in the rat / 대한치과교정학회지

The purpose of this study is to investigate the effect of loss of incisal function on the thickness, growth activities, ultrastructure of the condylar cartilage and on the muscle fibers of masseter superficialis, anterior belly of digastric muscle in the growing rats. 37 day-old-rats of which incisors had been trimmed every day received soft diet from weaning and were studied by the autoradiography, electron microscopy and muscle histochemistry. The results obtained were as follows: The thickness of the fibrous, proliferative layer in superior, posterosuperior portion of the condylar cartilage was significantly(p<0.01) reduced in experimental groups and the decrease rate of fibrous layer thickness was greater in posterosuperior portion than in superior portion of cartilage and was greater than in proliferative layer. In normal group, more cells of posterosuperior portion moved more rapidly towards the medullary cavity. In experimental group, the labelling index of posterosuperior portion was decreased in proliferative layer at 2 hours, in transitional layer at 1, 2 days, in hypertrophic layer at 4 days after injection relative to posterosuperior portion of control group. But labelling index of superior portion was not different from that of control group at all time course after injection. From the muscle histochemistry, the diameter of type IIB fibers in masseter superficialis muscle, type IIA, type IIB fibers in anterior belly of digastric muscle decreased significantly(p<0.01) relative to controls in experimental group. From electron microscopic study, in the fibrous layer of the posterosuperior portion of condylar cartilage in normal group, many fibroblast like cells near the joint cavity showed extensive remodelling activities in ultrastructure. There was no morphological changes between experimental and control group in all cartilage cell layers of superior portion but cells near the joint cavity in fibrous layer of posterosuperior portion of experimental group showed morphologically inactive state relative to control group.