Misexpression experiment of Tbx5 in axolotl (Ambystoma mexicanum) hindlimb blastema.

Axolotls (Ambystoma mexicanum) have the ability to regenerate amputated limbs throughout their life span. In the present study, we attempted to elucidate how axolotls can specify limb type correctly during the regeneration process. We misexpressed Tbx5 in regenerating hindlimb blastema, and consequently a forelimb-like hindlimb regenerated from the hindlimb blastema. On the other hand, no change was observed in Tbx5-overexpressing forelimb blastema, and thus we considered that Tbx5 plays a key role in the specification of forelimb during the regeneration process of axolotl limbs. However, axolotls' fore- and hindlimbs have very similar structures except for the number of fingers, and it was very difficult to judge whether the forelimb-like regenerate was a true forelimb or merely a forelimb-like hindlimb. Therefore, in order to confirm our conclusion, we have to investigate other genes that are expressed differentially between fore- and hindlimbs in future experiments.

Lmx-1b and Wnt-7a expression in axolotl limb during development and regeneration.

Axolotls (Ambystoma mexicanum) have the ability to regenerate amputated limbs throughout their life span. During limb regeneration as well as development, undifferentiated cells in the blastema acquire positional information to reproduce the original pattern along three cardinal limb axes: anteroposterior, proximodistal and dorsoventral. In the present study, we attempted to understand the molecular mechanism involved in patterning of axolotl limb development and regeneration along the dorsoventral (DV) axis. We cloned axolotl Lmx-1b and Wnt-7a, and investigated the expression pattern of these genes in developing and regenerating limbs. In axolotl, unlike in amniotes, Wnt-7a was expressed in a diffuse manner throughout both developing limb bud and regenerating limb blastema. Lmx-1b expression was observed at the dorsal mesenchyme in the developing and regenerating limbs. On the basis of the expression patterns of Lmx-1b and Wnt-7a, it was difficult to identify the interaction between these two genes as reported in amniotes in previous studies. Possibly, with regard to Lmx-1b expression, a Wnt-7a-independent mechanism may exist in axolotl limb development and regeneration.

Gross anatomical tractography (GAT) proposed a change from the 'Two laminae concept' to the 'Neuronal unit concept' on the structure of the human hippocampus.

The three dimensional structure of the human hippocampus was studied using the gross anatomical tractography (GAT) of Klingler technique. Eight hippocampi were obtained from seven donors to the Kanazawa Medical University, fixed in 5% formaldehyde, frozen and thawed twice, then dissected both by naked eye and under a dissecting microscope.The subiculum was segmented into 7-12 hillocks along the antero-posterior axis. The hillocks were organized with the white matter process and its gray matter covering. Cornu ammonis 3 (CA3) was represented by gray matter located in a longitudinal trough about 1 mm wide between the base of the fimbria and fiber bundles of the stratum lacunosum. CA3 was traversed and segmented by numerous short fiber fasciculi extending from the dentes of the margo denticulatus. The stratum radiatum, lacunosum and moleculare were differentially dissected. They not only contained systematically arranged neuronal fibers but also frameworks to allow passage of blood vessels. The polymorphic layer (PL) consisted of many italic L-shaped bars that were segmented, fused side-by-side and arrayed along the antero-posterior axis. The stratum granulosum (SG) lined the superior surface of PL as square plates and inferior surface of PL as thin folds. Thus, the SG was also segmented, although a little arbitrarily. CA4 was found not to be a neuronal plate, but instead comprised numerous neuronal rods that were segmentally arranged in accord with segmentation of CA3.On the basis of these segmentations, we conclude that, structurally, the human hippocampus is an antero-posterior succession of neuronal units, each consisting of the subicular hillock, dens, CA3, granular cell plates and folds, PL bars and CA4 rods.

The human hippocampus observed by scanning electron microscopy (SEM): the dentate gyrus is made of an array of the neuronal lamellae.

Scanning electron microscopy (SEM) was employed to clarify the three dimensional structure of the human hippocampus.The polymorphic layer was L-shaped in coronal histological sections. The superior limb and lateral two thirds of the inferior limb formed a continuous plate. This plate consisted of L-shaped bars that were fused side by side with borders that were, although incompletely, demarcated by the stratum granulosum. The medial one third of the inferior limb was independent part of these L-shaped bars and took part in formation of the dentes. There were 40 to 50 dentes, and each had segmental blood vessels. Thus, the polymorphic layer was organized on a segmental plan, 40- 50 in number, arrayed along the antero-posterior axis.CA4 was surrounded by the L-shaped polymorphic layer and also had the superior and inferior crura. The medial end of the inferior crus was enveloped by the medial one third of the polymorphic tissue and was completely independent from its neighbors. Therefore, CA4, too, may be segmentally constructed following the same plan as the bars of the polymorphic layer. These observations suggest that, first, three major components of the hippocampus, the stratum granulosum, polymorphic layer, and CA4, are constructed based on the same lamellar unit in the dentate gyrus, and, second, the individual lamellae appear as distinct bars in the medial one third, but form a plate in the lateral two thirds of these structures. There were 7 to 12 pyramidal hillocks, organized of the central process and its covering, in the subiculum. Pyramidal cells showed clear polarity in the hillock; the cell apex oriented to the central process and the base to the periphery. The axon emitted from the cell base and formed the alveus. Pyramidal hillocks caused slight waves of the stratum pyramidale on the lateral border of the hippocampus but did not affect the superior surface. Functional aspects of the segmental arrangement of neuronal units along the antero-posterior axis and their medio-lateral diversity were discussed in terms of the three-synapse pathway in the hippocampus.

Wound epithelium function in axolotl limb regeneration.

Axolotls (Ambystoma mexicanum) have the ability to regenerate amputated limbs. The amputation surface is promptly covered by wound epithelium (WE), which is significant for the initiation of limb regeneration. In the present study, we investigated the formation of functional WE by analyzing the migration of WE after amputation. In the center of the amputation surface, epithelial cells migrated from surrounding epidermis to form WE. Therefore, WE around the center of the amputation surface was composed of the cells with dorsal, ventral, anterior and posterior identities, and we tentatively called this WE with radial positional identities, "central WE". When regeneration was complete, central WE became the epidermis around the bifurcation between the first and second digits. In addition, when the artificial rotation of epidermis was performed before amputation, all examined limbs regenerated normally, and central WE formed the epidermis at the bifurcation between first and second digits, similarly to that in normal regeneration. On the basis of our observations, the most important factor for the initiation of regeneration is considered to be the discontinuity of positional identity existing in WE. It is possible that the location of bifurcation between first and second digits is specified by the positional discontinuity in WE.

Pitx1 expression in developing and regenerating axolotl limbs.

The expression of the homeobox transcription factor Pitx1 was investigated in the Mexican axolotl (Ambystoma mexicanum) during limb development and regeneration by whole-mount mRNA in situ hybridizations. This clone shares high amino acid identity with Pitx1 from other vertebrates (92% Xenopus; 87% chick; 75% human and mouse) within the region isolated. In the developing limbs, Pitx1 was expressed in hindlimb mesenchyme, as has been reported in other species. The expression pattern in the hindlimb might have been conserved during evolution. In the regenerating limbs, Pitx1 was expressed in both fore- and hindlimb blastemas. Our observations suggest two roles of Pitx1 in the axolotl: one is to determine the hindlimb pattern during development, and the other that relates to proliferation of regenerating tissues without regard to fore- or hindlimb.

Fiber dissection technique for demonstrating the lateral lemniscus of the human brain.

This report describes fiber dissection technique for tracing the auditory pathway from the cochlear nerve to the medial geniculate body via the lateral lemniscus, inferior colliculus and inferior brachium. Some fibers of the lateral leminiscus appear to reach the thalamus in conjunction with fibers of the medial lemniscus.

Proliferating cells in the rat anterior pituitary during the postnatal period: immunoelectron microscopic observations using monoclonal anti-bromodeoxyuridine antibody.

Proliferating cells in the male rat anterior pituitary at 1, 3, 5, and 8 weeks of age were labeled with bromodeoxyuridine (BrdU) and studied by light and electron microscopic immunocytochemistry using anti-BrdU. They decreased in number from 402+/-31/mm(2) at 1 week to 50+/-1.5/mm(2) at 8 weeks, while their cell area increased by about twofold during this period. They had a slightly higher nucleus/whole cell (N/C) ratio than non-proliferating cells. According to their ultrastructure we classified them into granular and agranular cells. The percentage of granular cells ranged from 73% to 82% of all the proliferating cells during the period studied. They had many granules of various sizes and shapes, and some contained growth hormone and prolactin. Agranular cells, constituting 18-27% of proliferating cells, were small and had a high N/C ratio, indicating their immaturity. Moreover, they showed several features of folliculo-stellate (FS) cells: they showed no secretory granules in the cytoplasm, extended thin cytoplasmic processes, and sometimes they constructed a follicle among them. These results suggest: (1) the majority of proliferating cells were mature cells producing anterior pituitary hormone(s) and (2) most of the agranular proliferating cells maybe FS cells. The possibility of the latter is discussed.

The distribution and morphology of lymphatic vessels on the peritoneal surface of the adult human diaphragm, as revealed by an ink-absorption method.

Application of india ink to the peritoneal and pleural surfaces of the adult human diaphragm allowed visualization of the distribution and morphology of the lymphatic vessels by light microscopy and scanning electron microscopy. The diaphragms examined had been fixed and stored in 10% formalin. Numerous lymphatic vessels were stained black with india ink, presenting reticular, radial-meshwork, ladder-like and lacy patterns. They were distributed throughout the entire sternocostal part. Analysis by light and scanning electron microscopy of the areas indicated by india ink revealed the presence of primary lymphatic vessels that formed lymphatic lacunae and stomatal openings to the peritoneal cavity. A layer of secondary collecting lymphatic vessels was located cranially with respect to the layer of primary lymphatic vessels. Thus, the peritoneum had at least two layers of lymphatic vessels. These lymphatic vessels were not tubular vessels but resembled flat cisternae, as has been suggested in the case of the mouse diaphragm. The pleura lacked lymphatic stomata and had no such double-layered lymphatic organization. This is the first report that showed distribution and morphology of the lymphatic vessels in the diaphragmatic peritoneum of the formalin-fixed, adult human diaphragm. The method and results in the present study may contribute to morphological analysis of the lymphatic system in the wall of the human body cavity.

Proliferation and differentiation of rat anterior pituitary cells.

Studies on the proliferation and differentiation of the cells in the rat anterior pituitary were reviewed. The mitotic rate of anterior pituitary is low in the control adult animal, but it increased by stimulation, such as by ablation of the target organ. A high mitotic rate was also reported during ontogenesis of the pituitary. Concomitant with this augmented mitosis, the number of those cells that are double-labeled with the marker of proliferation and the antibody to pituitary hormones increased as well. The percentage of these double-labeled cells in all the proliferating cells is less than 10%, suggesting that about 1/10 of the proliferating cells are involved in producing pituitary cells. This percentage for GH cells is 30-40% at most, suggesting very active production of them. The percentage of the double-labeled cell in all the hormone-producing cells is within 10% in all cell-types of the pituitary, including GH cells. When the proliferation is detected by a more sensitive method, this percentage increased to 20-40%, suggesting that the self-mitosis of the pituitary cells contributes considerably to their proliferation at a certain period during their ontogenesis.

Mitoses of thyrotrophs contribute to the proliferation of the rat pituitary gland during the early postnatal period.

Development of thyrotrophs in the rat pituitary at 3, 7 and 10 days after birth was quantitatively studied by labelling the proliferating cells with bromodeoxyuridine (BrdU) and with the proliferating cell nuclear antigen (PCNA), and the immunostaining of thyrotrophs was applied to the same tissue section. Double administration of BrdU at 9:00 h and 19:00 h, increased the numerical volume density (Nv) of labelled cells by about 1.5-fold of that obtained with a single injection at 9:00 h. When PCNA was used to determine hyperplasia, the Nv of labelled cells further increased greatly. In accordance with these results, the Nv of the thyrotrophs that were also labelled with BrdU or PCNA increased likewise. These cells comprised 3-7% of all BrdU- or PCNA-labelled cells, indicating that about 1/20 of the proliferating cells are involved in producing new thyrotrophs. On the other hand, their percentage in all thyrotrophs was 8.8%, 18.4% or 38.7% in 3-day neonates with single or double BrdU injections, or when PCNA was detected. These high percentages indicate a considerable contribution by the mitosis of already existing thyrotrophs to their proliferation in the early postnatal period.

Relative positions of the arteries and veins on the dorsolateral surface of the human cerebrum.

We investigated whether arteries pass superficial to veins or whether veins pass superficial to arteries at artery-vein crossings on the anterior, dorsolateral, and posterior surfaces of the human cerebrum. We examined a total of 2,266 artery-vein crossings on 40 sides of 20 cadavers. At 2,059 crossings (91%), the vein passed superficial to the artery. Thus, vein (V), artery (A), and nerve (N), if we regard the cerebrum as nerve, were generally arranged in the order VAN from the superficial to the deep layers. This concept is important for a positional understanding of blood vessels on the cerebrum and it is useful for the understanding of fluid-drainage pathways from the cerebral cortex in various pathological conditions.