Double-layered two-directional somatopleural cell migration during chicken body wall development revealed with local fluorescent tissue labeling.

The thoracic ventral body wall consists of the rib, the sternum, the intercostal muscles, and the connective tissues surrounding them. The ribs and the intercostal muscles are derived from the somite. The connective tissues are derived from the somatic layer of the lateral plate mesoderm, somatopleure. The lateral growth of the somatopleure forms the primary ventral body wall. The migration of somitic cells into the somatopleure generates the secondary body wall. As the migrating behavior of the somatopleural cells during secondary body wall formation is still unclear, we investigate here the migratory behavior of the somatopleural cells in the thorax during chicken ventral body wall development by labeling the thoracic somatopleural cells one-somite-wide by DiI labeling or gene transfection of the enhanced green fluorescent protein and observe their distribution assisted by the tissue-clearing technique FRUIT. Our labeling experiments revealed the rostral migration of the somatopleural cells into a deep part of the thoracic body wall in embryonic day 6.5 chickens. For embryonic day 8.5 chickens, these deep-migrating somatopleural cells were found around the sternal ribs. Thus, we identified the double-layered two-directional migrating pathways of the somatopleural cells: the rostral migration of the deep somatopleural cells and the lateral migration of the superficial somatopleural cells. Our findings imply that the rostral migration of deep somatopleural cells and the lateral migration of superficial ones might be associated with the developing sternal ribs and the innervation of the thoracic cutaneous nerves, respectively.

Morphological Classification of the Medial Frontal Cortex Based on Cadaver Dissections: A Guide for Interhemispheric Approach.

The medial frontal cortex (MFC) is a part of the medial surface of the frontal lobe situated in the rostral portion of the corpus callosum (CC). In a surgical interhemispheric approach (IHA), the MFC covers the anterior communicating artery (Aco) complex until the final stage of dissection. To clarify the anatomical relationship between the MFC and the Aco complex, and to facilitate orientation in IHA, we analyzed the morphological features of the MFC in number, size, and pattern of gyri from the medial surface of the hemisphere in the subcallosal portion using 53 adult cadaveric hemispheres. The mean width of the MFC excluding cingulate gyrus (MFCexcg) was 20.6 ± as mm in the subcallosal portion. MFCexcg consisting of 2, 3, 4, or 5 gyri were observed in 7.5%, 56.6%, 32.1%, or 3.8% of the hemispheres, respectively. Bilateral MFCexcg consisting of >2 gyri were observed in approximately 85% of the hemispheres. Therefore, in many cases, the dissection performed at 2 cm upward from the base of the straight gyrus (SG) or 3-4 gyri of the MFC is sufficient to safely reach the upper portion of the cistern of lamina terminalis located distal to the Aco complex in IHA. The MFC is a good landmark for intraoperative orientation in IHA.

Demonstration of Human Fetal Bone Morphology with MR Imaging: A Preliminary Study.

PURPOSE: CT is a useful modality for the evaluation of fetal skeletal dysplasia but radiation exposure is unavoidable. The purpose of this study is to compare the usefulness of MRI and CT for evaluating the fetal skeletal shape. METHODS: This study was approved by our Institutional Review Board. Fetal specimens (n = 14) were scanned on a 3T MRI scanner using our newly-developed sequence. It is based on T2*-weighted imaging (TR, 12 ms; TE for opposed-phase imaging, 6.1 ms, for in-phase imaging, 7.3 ms; flip angle, 40°). The specimens were also scanned on a 320 detector-row CT scanner. Four radiologists visually graded and compared the visibility of the bone shape of eight regions on MRI- and CT-scans using a 5-point grading system. RESULTS: The diagnostic ability of MRI with respect to the 5th metacarpals, femur, fibula, and pelvis was superior to CT (all, P < 0.050); there was no significant difference in the evaluation results of observers with respect to the cervical and lumbar spine, and the 5th metatarsal (0.058 ≤ P ≤ 1.000). However, the diagnostic ability of MRI was significantly inferior to CT for the assessment of the bone shape of the thoracic spine (observers A and C: P = 0.002, observers B and D: P = 0.001). CONCLUSION: The MRI method we developed represents a potential alternative to CT imaging for the evaluation of the fetal bone structure.

An in vitro model of region-specific rib formation in chick axial skeleton: Intercellular interaction between somite and lateral plate cells.

The axial skeleton is divided into different regions based on its morphological features. In particular, in birds and mammals, ribs are present only in the thoracic region. The axial skeleton is derived from a series of somites. In the thoracic region of the axial skeleton, descendants of somites coherently penetrate into the somatic mesoderm to form ribs. In regions other than the thoracic, descendants of somites do not penetrate the somatic lateral plate mesoderm. We performed live-cell time-lapse imaging to investigate the difference in the migration of a somite cell after contact with the somatic lateral plate mesoderm obtained from different regions of anterior-posterior axis in vitro on cytophilic narrow paths. We found that a thoracic somite cell continues to migrate after contact with the thoracic somatic lateral plate mesoderm, whereas it ceases migration after contact with the lumbar somatic lateral plate mesoderm. This suggests that cell-cell interaction works as an important guidance cue that regulates migration of somite cells. We surmise that the thoracic somatic lateral plate mesoderm exhibits region-specific competence to allow penetration of somite cells, whereas the lumbosacral somatic lateral plate mesoderm repels somite cells by contact inhibition of locomotion. The differences in the behavior of the somatic lateral plate mesoderm toward somite cells may confirm the distinction between different regions of the axial skeleton.

Morphological Pattern and Classification of the Superficial Middle Cerebral Vein by Cadaver Dissections: An Embryological Viewpoint.

In this study, we used 45 adult cadaveric cerebral hemispheres to investigate the anatomical classification of the superficial middle cerebral vein (SMCV) based on the number of stems, course, and anastomosis at the distal portion. We classified the SMCVs into five types based on embryological concept. Type A (18 cases, 40.0%) is that the frontosylvian veins (FSVs) merge with the vein of Trolard (VT) and the vein of Labbé (VL) at the distal portion of the sylvian fissure. Type B (5 cases, 11.1%) is that the temporosylvian veins (TSVs) merge with the VT and the VL at the distal portion. Type C (13 cases, 28.9%) is that no vein merge with the VT and the VL at the distal portion. The VT merges with the SMCV from the FSV and the VL merges with the SMCV from the TSV. They course along the sylvian fissure and merge at the proximal portion. In Type D (eight cases: 17.8%), the VT and the VL merge at the distal portion, and the SMCV from the FSV and the SMCV from the TSV join their confluence without merging. Type E (one case, 2.2%) show an undeveloped SMCV. Formation rate of intravenous anastomoses or bridging veins(BVs) at the distal portion between the frontosylvian trunk (FST) and the temporosylvian trunk (TST), between the FST and the temporal lobe, and between the TST and the frontal lobe was very low, because these formation may be difficult to occur during the embryological process in which the SMCV is formed from the telencephalic vein.

From the primitive streak to the somitic mesoderm: labeling the early stages of chick embryos using EGFP transfection.

Mesoderm is derived from the primitive streak. The rostral region of the primitive streak forms the somitic mesoderm. We have previously shown the developmental origin of each level of the somitic mesoderm using DiI fluorescence labeling of the primitive streak. We found that the more caudal segments were derived from the primitive streak during the later developmental stages. DiI labeled several pairs of somites and showed the distinct rostral boundary; however, the fluorescence gradually disappeared in the caudal region. This finding can be explained in two ways: the primitive streak at a specific developmental stage is primordial of only a certain number of pairs of somites, or the DiI fluorescent dye was gradually diluted within the primitive streak by cell division. Here, we traced the development of the primitive streak cells using enhanced green fluorescent protein (EGFP) transfection. We confirmed that, the later the EGFP transfection stage, the more caudal the somites labeled. Different from DiI labeling, EGFP transfection performed at any developmental stage labeled the entire somitic mesoderm from the anterior boundary to the tail bud in 4.5-day-old embryos. Furthermore, the secondary neural tube was also labeled, suggesting that not only the somite precursor cells but also the axial stem cells were labeled.

Radiation dose reduction at MDCT with iterative reconstruction for prenatal diagnosis of skeletal dysplasia: preliminary study using normal fetal specimens.

OBJECTIVE: The purpose of this study was to investigate to what degree the radiation dose can be reduced without affecting the ability to evaluate normal fetal bones at MDCT with iterative reconstruction. MATERIALS AND METHODS: Fifteen normal fetal specimens immersed in containers (30- and 35-cm diameter) were scanned with a 64-MDCT scanner, with tube voltage of 100 kVp and tube current of 600, 300, 150, 100, and 50 mA. Images were subjected to adaptive statistical iterative reconstruction (ASIR). The fetal dose was measured using glass dosimeters. We calculated the relative ratio of the dose at 600 mA. Image quality was evaluated on maximum-intensity-projection and volume-rendering images. Two radiologists recorded the visualization scores of five regions. Images at 600 mA were considered to be standard. RESULTS: With the 30-cm-diameter container, the fetal dose was 10.15 mGy (relative ratio, 100%) at a tube current of 600, 51% at 300, 25% at 150, 17% at 100, and 9% at 50 mA. With the 35-cm-diameter container the fetal dose was 10.01 mGy (relative ratio, 100%) at 600, 47% at 300, 24% at 150, 17% at 100, and 8% at 50 mA. Visual evaluation showed that in both containers, with ASIR 90%, there was a statistically significant difference between 50-and 600-mA images (p<0.01) but not between 600-mA images and those acquired at 100, 150, and 300 mA (p=0.08-1.00). CONCLUSION: The fetal radiation dose for the evaluation of normal fetal bones can be reduced by 83% with ASIR 90%.

Potential risks of femoral tunnel drilling through the far anteromedial portal: a cadaveric study.

PURPOSE: The purpose of this study was to estimate the potential risks when drilling femoral tunnels through the far anteromedial portal in double-bundle anterior cruciate ligament reconstruction in cadaveric knees. METHODS: Ten cadaveric knees were used. We drilled the anteromedial bundle (AMB) and posterolateral bundle (PLB) through the far anteromedial portal at 3 different knee flexion angles: 70 degrees, 90 degrees, and 110 degrees. We measured the shortest distance to the common peroneal nerve and the posterior articular cartilage of the lateral femoral condyle and the femoral tunnel length. RESULTS: At 70 degrees, the distance to the nerve was less than 10 mm in 7 AMB cases and in 9 PLB cases, and the distance to the cartilage was less than 10 mm in all the AMB and PLB cases. At 90 degrees, the distance to the nerve was less than 10 mm in 1 AMB and 5 PLBs, and the distance to the cartilage was less than 10 mm in 2 AMBs and all the PLBs. On the other hand, at 110 degrees , the distance to the nerve was greater than 10 mm in all the AMBs and PLBs, and the distance to the cartilage did not exceed 10 mm in just 2 of the PLBs. CONCLUSIONS: In our cadaveric study we found that the low knee flexion angles when drilling femoral tunnels through the far anteromedial portal might have the potential risks of damage to the common peroneal nerve and the posterior articular cartilage, and the risks would be decreased at higher degrees of knee flexion. However, we found there was a 20% risk of damage to the cartilage while drilling the PLB at 110 degrees. CLINICAL RELEVANCE: High knee flexion angles are recommended to avoid damage to the nerve and the cartilage when drilling femoral tunnels through the far anteromedial portal in double-bundle anterior cruciate ligament reconstruction.

Elevated intraocular pressure, optic nerve atrophy, and impaired retinal development in ODAG transgenic mice.

PURPOSE: In an earlier study, a cDNA was cloned that showed abundant expression in the eye at postnatal day (P)2 but was downregulated at P10; it was named ODAG (ocular development-associated gene). Its biological function was examined by generating and analyzing transgenic mice overexpressing ODAG (ODAG Tg) in the eye and by identifying ODAG-binding proteins. METHODS: Transgenic mice were generated by using the mouse Crx promoter. EGFP was designed to be coexpressed with transgenic ODAG, to identify transgene-expressing cells. Overexpression of ODAG was confirmed by Northern and Western blot analysis. IOP was measured with a microneedle technique. The eyes were macroscopically examined and histologically analyzed. EGFP expression was detected by confocal microscope. Proteins associated with ODAG were isolated by pull-down assay in conjugation with mass spectrometry. RESULTS: Macroscopically, ODAG Tg exhibited gradual protrusion of the eyeballs. The mean IOP of ODAG Tg was significantly higher than that of wild-type (WT) littermates. Histologic analysis exhibited optic nerve atrophy and impaired retinal development in the ODAG Tg eye. EGFP was expressed highly in the presumptive outer nuclear layer and weakly in the presumptive inner nuclear layer in the ODAG Tg retina. Rab6-GTPase-activating protein (Rab6-GAP) and its substrate, Rab6, were identified as ODAG-binding proteins. CONCLUSIONS: Deregulated expression of ODAG in the eye induces elevated intraocular pressure and optic nerve atrophy and impairs retinal development, possibly by interfering with the Rab6/Rab6-GAP-mediated signaling pathway. These results provide new insights into the mechanisms regulating ocular development, and ODAG Tg would be a novel animal model for human diseases caused by ocular hypertension.

Body wall morphogenesis: limb-genesis interferes with body wall-genesis via its influence on the abaxial somite derivatives.

The vertebrate body wall is regionalized into thoracic and lumbosacral/abdominal regions that differ in their morphology and developmental origin. The thoracic body wall has ribs and intercostal muscles, which develops from thoracic somites, whereas the abdominal wall has abdominal muscles, which develops from lumbosacral somites without ribs cage. To examine whether limb-genesis interferes with body wall-genesis, and to test the possibility that limb generation leads to the regional differentiation, an ectopic limb was induced in the thoracic region by transplanting prospective limb somatopleural mesoderm of Japanese quail between the ectoderm and somatopleural mesoderm of the chick prospective thoracic region. This ectopic limb generation induced the somitic cells to migrate into the ectopic limb mesenchyme to become its muscles and caused the loss of distal thoracic body wall (sterno-distal rib and distal intercostal muscle), without causing any significant effect on the more proximal region (proximal rib, vertebro-distal rib and proximal intercostal muscle). According to a new primaxial-abaxial classification, the proximal region is classified as primaxial and the distal region, as well as limb, is classified as abaxial. We demonstrated that ectopic limb development interfered with body wall development via its influence on the abaxial somite derivatives. The present study supports the idea that the somitic cells give rise to the primaxial derivatives keeping their own identity and fate, whereas they produce the abaxial derivatives responding to the lateral plate mesoderm.

Pigment epithelium-derived factor promotes neurite outgrowth of retinal cells.

The ability of pigment epithelium-derived factor (PEDF) to promote neurite outgrowth of retinal cells through mitogen-activated protein kinase (MAPK) pathways was examined. Neurite outgrowth effects of PEDF were determined by quantifying the neurite length extending from cultured chick embryo retinal explants, and neurite outgrowth ratio of R28 cells (a neural cell line derived from the neonatal rat retina). MAPK activity levels were determined by inhibition assays. The contribution of signaling pathway was quantified with a specific inhibitor for MAPK: PD98059. PEDF (50 ng/ml) promoted chick retinal neurite elongation and increased the extent of R28 cell neurite outgrowth. PD98059 decreased neurite elongation of chicken retinal explants and the extent of R28 cell neurite outgrowth. PEDF possibly promotes neurite outgrowth for retinal cells by activating MAPK pathways. These data suggest that PEDF provides a useful support for retinal cells through the MAPK pathway and leads to the progress of therapy for many retinal diseases.

Three developmental compartments involved in rib formation.

When the thoracic somitic mesoderm was separated from the neural tube and the notochord with a piece of aluminum foil in two-day chick embryos, seven days after the operation ribs lacked their proximal part. The embryos were rescued by co-transplanting the notochord, the ventral half of neural tube, or QT6 cells transformed with Shh, on the somite side of the aluminum foil insert. Thus, proximal rib development depends on the notochord and the ventral neural tube, an effect which might be mediated through Shh secreted by these axial tissues. On the other hand, when the thoracic somitic mesoderm was separated from the surface ectoderm by a piece of polyethylene terephthalate film, the distal parts of the ribs were missing, suggesting that distal rib development depends on surface ectoderm. In these embryos, expression of Pax3 was weak and perturbed showing that the dermomyotome developed abnormally. It is not clear whether the development of distal rib is mediated by the dermomyotome, or the ectoderm. It has previously been shown that sternal rib development depends on lateral plate mesoderm. As to the distal rib, it is considered to be composed of two parts. Thus, the rib is composed of three developmental compartments, in agreement with a recently presented classification of somite derivatives as primaxial and abaxial.

Somite development without influence of the surface ectoderm in the chick embryo: the compartments of a somite responsible for distal rib development.

In the development of the somite, signals from neighboring tissues have been suggested to play critical roles. We have found that when interaction between the ectoderm and the somite is blocked by inserting a piece of polyethylene terephatalate film between them in 2-day-chicken embryo, one of the derivatives of somite, the distal rib, did not form. We examined somite development after the operation, to know the correlation between somite development and distal rib formation. In the operated embryo, the dermomyotome was medio-laterally shorter than in the normal embryo, and Pax3 and Sim1 expressions that are seen in the lateral part of normal dermomyotomes were not found, suggesting that the lateral part of the dermomyotome was missing. Although the sclerotome appeared to be normal in its histology and Pax1 expression pattern in the operated embryo, we could not detect the expression of either Scleraxis nor gamma-FBP that are expressed in the cells around the boundaries between the adjacent dermomyotomes in normal embryos. Thus, under the influence of surface ectoderm, the lateral part of dermomyotome and/or the mesenchyme around rostral and caudal edges of dermomyotomes are suggested to play an important role in the distal rib development.

Formaldehyde concentration in the air and in cadavers at the gross anatomy laboratory in Hiroshima University.

The formaldehyde concentration in the air and in various tissues of 35 human cadavers were measured during a gross anatomy course held at the Faculty of Medicine of Hiroshima University in the 2003 educational year. Atmospheric formaldehyde levels were 0.25-0.55 ppm and thus less than the upper limit of the guideline for formaldehyde exposure (0.5 ppm) set by the Japan Society for Occupational Health (1988) except for one out of 10 measurements. The formaldehyde concentrations in tissues were as follows: the lung, 0.12 +/- 0.09% (n=29); the liver, 0.12 +/- 0.09% (n=29); and the brachioradialis muscle, 0.11 +/- 0.09% (n=30). Considerable variation was found among the cadavers and these values were lower than those of Tsurumi University which provided the only other data (average formaldehyde concentrations ranged from 0.27 to 0.32%). At Hiroshima University, blood is allowed to drain during embalming, whereas it is not at Tsurumi University. Differences in the embalming procedure are thus responsible for low and fluctuating formaldehyde concentrations in cadavers at Hiroshima University, and it is conceivable that relatively low formaldehyde levels in the air result from low formaldehyde concentrations in cadavers and good room ventilation (10 room-air changes per hour). However, the Japanese Ministry of Health and Welfare recommended lower formaldehyde exposure levels (0.08 or 0.25 ppm) in 2002. Thus, it may be necessary to further reduce formaldehyde levels in the gross anatomy laboratory by means of such measures as neutralizing formaldehyde with ammonium carbonate; using a locally ventilated dissection work-table, etc.

Developmental Plasticity of the Prospective Dermatome and the Prospective Sclerotome Region of an Avian Somite: (somite/dermomyotome/sclerotome/determination/quail-chick chimera).

The ventro-medial wall of a somite gives rise to the sclerotome and then to cartilaginous axial skeleton, while the dorso-lateral wall differentiates into the dermomyotome to form dermal mesenchyme and muscle. Although previous studies suggested pluri-potency of somite cell differentiation, apparent pluri-potency may be the result of migration of predetermined cells. To investigate whether the developmental fate of any region is determined, I isolated fragments of a region of a quail somite and transplanted them into chick embryos. When a fragment of the ventral wall of a quail somite, the prospective sclerotome, was transplanted into a chick embryo between the ectoderm and a newly formed somite, the transplanted quail cells were shown to form myotome and mesenchyme in 4-day chimera embryos and to form muscle and dermal tissue in 9-day chimeras. On the other hand, when a fragment of the dorsal wall of a quail somite, the prospective dermomyotome, was transplanted into a chick embryo between the neural tube and a newly formed somite, the graft gave rise to mesenchyme around the neural tube and notochord and then to vertebral cartilage. Thus the developmental fate of a region of a somite was shown not to be determined at the time of somite segmentation, confirming previous observations.

Regulation of Cell Number in Formation of the Dorsal Root Ganglion Revealed by Transplantation of Quail Neural Crest Cells into Chick Embryos: (dorsal root ganglia/cell number/chick-quail chimera/neural crest/regulation).

Neural crest cells appear transiently in early embryogenesis on the dorsal surface of the neural tube and subsequently migrate along specific pathways. Some migrate to between the neural tube and somites, aggregating to form the rudiments of dorsal root ganglia (DRG). The size of DRG at a given somite level is almost constant in all chick embryos. To determine the mechanisms controlling the size of DRG, we transplanted neural crest cells of 2.5-day-old quail embryos into 2.5-day-old chick embryos between the neural tube and the somites, and examined the size of DRG in these chimeric embryos with extra neural crest cells 2 days after the operation, when natural cell death in DRG had not yet occurred. The DRG on the operated side were composed of both chick and quail cells in various proportions. The cell numbers of these chimeric DRG were almost the same as those of the normal DRG on the opposite side. That is, there were significantly fewer chick cells in chimeric DRG than in DRG composed of only chick cells on the opposite unoperated side. This finding indicates that the size of DRG is not determined in migrating neural crest cells but is regulated by the circumstances.

Do Peanut Agglutinin Receptors on Somites Control the Behavior of Neural Cells?: (PNA receptors/somite/resegmentation/rostro-caudal axis/gangliogenesis).

Peanut agglutinin (PNA) receptors are expressed in the caudal halves of sclerotomes in chick embryos after 3 days of incubation (stages 19-20 of Hamburger & Hamilton). The neural crest cells forming dorsal root ganglia (DRG) and motor nerves appear to avoid PNA positive regions and concentrate into rostral halves of sclerotomes. To investigate the role of PNA receptors in gangliogenesis and nerve growth, we examined PNA binding ability in quail sclerotomes and in chick-quail chimeric embryos made by transplanting quail somites to chick embryos, comparing the development of DRG, motor nerves and sclerotomes. PNA did not bind to any part of the somites of 4.5-day quail embryos, although dorsal root ganglia and motor nerves appeared only in the rostral halves of sclerotomes as in chick embryos. Moreover, in spite of no PNA binding ability of the transplanted quail somite in 4.5-day chick-quail chimeric embryos, DRG and motor nerves derived from chick tissues appeared only in the rostral halves of the sclerotomes derived from these somites. Thus, both quail and chick neural crest cells and motor nerves recognized the difference between the rostral and caudal halves of sclerotomes of quail embryos in the absence of PNA binding ability, indicating that PNA binding site on somite cells does not support the selective neural crest migration and nerve growth.

DIFFERENTIATION AND DEDIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN SHORT- AND LONG-TERM CULTURES.

The crystallin synthesis of rat lens cells in cell culture systems was studied in relevance to their terminal differentiation into lens fibers. SDS-gel electrophoresis combined with several immunological techniques showed that γ-crystallin is a fiber-specific lens protein and is not localized in the epithelium of either newborn or adult lenses. When lens epithelial cells of newborn rats were cultured in vitro, α-crystaIlin was detected in many, but not all, of cells cultured for 10 days. Cells with α-crystallin gradually changed their shape into a flattened filmy form and finally differentiated into lentoid bodies. The differentiation of lentoid bodies was also found in cultures of epithelial cells obtained from adult lenses. The molecular constitution of lentoid bodies was the same as that of lens fibers in situ. The differentiation of lentoid bodies occurred successively for 5 months in cultures of lens epithelial cells. Most of the proliferating cells, however, lost α-crystallin during the culture period. Thereafter, they did not show any sign of further differentiation into lens fibers. Four clonal lines were established from these cells. One protein which is specific to the lens epithelium and the neural retina in situ (tentatively named as ßu -crystallin) was maintained in all lines, suggesting that some specific properties of ocular cells remain in the lined cells.