Maki Yuguchi

Development of the Tarsometatarsal Skeleton by the Lateral Fusion of Three Cylindrical Periosteal Bones in the Chick Embryo (Gallus gallus)

An avian tarsometatarsal (TMT) skeleton spanning from the base of toes to the intertarsal joint is a compound bone developed by elongation and lateral fusion of three cylindrical periosteal bones. Ontogenetic development of the TMT skeleton is likely to recapitulate the changes occurred during evolution but so far has received less attention. In this study, its development has been examined morphologically and histologically in the chick, Gallus gallus. Three metatarsal cartilage rods radiating distally earlier in development became aligned parallel to each other by embryonic day 8 (ED8). Calcification initiated at ED8 in the midshaft of cartilage propagated cylindrically along its surface. Coordinated radial growth by fabricating bony struts and trabeculae resulted in the formation of three independent bone cylinders, which further became closely apposed with each other by ED13 when the periosteum began to fuse in a back‐to‐back orientation. Bone microstructure, especially orientation of intertrabecular channels in which blood vasculature resides, appeared related to the observed rapid longitudinal growth. Differential radial growth was considered to delineate eventual surface configurations of a compound TMT bone, but its morphogenesis preceded the fusion of bone cylinders. Bony trabeculae connecting adjacent cylinders emerged first at ED17 in the dorsal and ventral quarters of intervening tissue at the mid‐diaphyseal level. Posthatch TMT skeleton had a seemingly uniform mid‐diaphysis, although the septa persisted between original marrow cavities. These findings provide morphological and histological bases for further cellular and molecular studies on this developmental process. Anat Rec 293:1527–1535, 2010.

Characterization of mesenchymal progenitor cells in the crown and root pulp of primary teeth

The existence of progenitor/mesenchymal stem cells (MSCs) was demonstrated previously in human primary/deciduous teeth. In this study, we examined dental pulp cells from root portion (root cells) of primary teeth without discernible root resorption and compared them with pulp cells from the crown portion (crown cells). Root cells and crown cells were characterized and compared to each other based on progenitor/MSC characteristics and on their generation efficiency of induced pluripotent stem (iPS) cells. Root cells and crown cells included cells manifesting typical progenitor/MSC properties such as osteogenic and adipogenic differentiation potential and clonogenicity. Interestingly, root cells showed a higher expression level of embryonic stem cell marker, KLF4, than crown cells. Moreover, the number of colony-forming unit-fibroblast and cell proliferation rate were higher for root cells than crown cells, and the efficiency of generating iPS cells from root cells was approximately four times higher than that from crown cells. Taken together, these results suggest that root cells from primary teeth show the MSC-like properties and thus could be a potent alternative source for iPS cell generation and the subsequent transplantation therapy.

Development of collagen fibres and lysyl oxidase expression in the presumptive dermis of chick limb bud.

With 4 figures and 1 table

Whole-mount bone and cartilage staining of chick embryos with minimal decalcification

Abstract Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.

Late Deposition of Elastin to Vertical Microfibrillar Fibers in the Presumptive Dermis of the Chick Embryonic Tarsometatarsus

Fibrillin microfibrils are integral components of elastic fibers and serve as a scaffold for elastin deposition. However, microfibrillar fibers (MFs) are not necessarily committed to develop into so‐called elastic fibers. In dermis, elastin‐free oxytalan MFs originating from the dermoepidermal junction are continuous to elaunin‐type MFs (with a small amount of elastin) in the deeper papillary dermis, whereas the reticular dermis contains elastic fibers, or MFs embedded largely in elastin. In this study, we have investigated temporospatial patterns of elastin deposition on the MFs in tarsometatarsal presumptive dermis. While the earliest expression of elastin was demonstrated immunohistochemically as early as embryonic day 4 (ED4) in the wall of cardiac outflow and pharyngeal arch arteries, its deposition in the tarsometatarsus was first detected at ED6 in the deeper mesenchyme and at ED13 in the subectodermal mesenchyme. In the latter tissue, MFs had been organized perpendicularly to the covering ectoderm by ED4, well before an overt accumulation of collagenous matrix. Elastin deposition was observed initially in a punctate manner at ED13 and afterward became continuous along MFs. However, a characteristic spaced array of subectodermal vertical MFs was disorganized by ED17. These findings suggest that elastin deposition in the subectodermal MFs is not deployed by continuous, orderly propagation from elastic fibers in the deeper mesenchyme but occurs de novo in multiple foci along vertical MFs. Moreover, the present chronology of elastin deposition indicates that subectodermal, elastin‐free MFs function as a transient, but primary fibrous structure in the presumptive dermis before the accumulation of collagenous matrix. Anat Rec, 290:1300‐1308, 2007.

Progressive Bundling of Fibrillin Microfibrils into Oxytalan Fibers in the Chick Presumptive Dermis

Dorsoventral fibers in the presumptive dermis of the chick limb bud reported first by Hurles group in 1989 are now revealed as bundles of fibrillin microfibrils (Isokawa et al., 2004). The bundles, which could be called oxytalan fibers at the light microscopic level, are aligned perpendicularly to the overlying ectoderm and form a unique fiber array, originating directly from the basal lamina. This well‐oriented organization is beneficial in examining the process of in vivo bundling of microfibrils into oxytalan fibers. In this study, sections through the presumptive limb dermis were preferentially prepared from chick embryos at Days 4–6 (ED4‐6). Immunohistochemically, fibrillin‐positive dots representing cross‐sectioned surfaces of individual fibers, increased in size from ED4 to 6, but their number per unit area remained constant. Ultrastructurally, a single oxytalan fiber at ED4 consisted of ∼15 microfibrils; the latter number increased fourfold from ED4 to 5 and threefold from ED5 to 6. Oxytalan fibers were all closely associated with mesenchymal cell; notably, the fibers at ED5 and 6 were held in a shallow ditch on the cell body or by lamellipodial cytoplasmic protrusion. In the sites of cell–fiber adhesion, microfibrils in the periphery of an oxytalan fiber appeared to adhere directly or by means of short flocculent strands to a nearby cell membrane; the latter showed a thickening of plasmalemma and its undercoat, indicating the presence of adhesive membrane specification. These findings suggest that the bundling of microfibrils is a progressive and closely cell‐associated process. Anat Rec, 2013.

Highly reproducible skeletal deformities induced by administration of β-aminopropionitrile to developing chick embryos.

The formation of cross-linkages between and within collagen is catalyzed by lysyl oxidase, which can be inhibited by β-aminopropionitrile (BAPN), a lathyrogen from sweet pea (Lathyrus odoratus) seeds. The quality and integrity of the collagenous template of skeletal elements depend on an appropriate concentration of collagen cross-links. In this study, chick embryos treated in ovo with BAPN on embryonic days (ED) 4-9 were found to develop multiple skeletal deformities. The most readily discernible and highly reproducible deformity was evident in the tibiotarsus, on which we focused to explore the chronology of the malformation process. Several lines of observation indicated that the bending deformity observable at ED10 in the tibiotarsus was inducible by BAPN administered on ED4-8; in other words, administration of BAPN on ED8 was sufficient to induce the deformity by ED10, whereas administration on ED9 was ineffective. Ultrastructurally, osteoclasts appeared to show enhanced activity in the medullary surface of the bone collar after BAPN administration. In addition, bone hyperplasia associated with the bending deformity was suggested to be correlated with higher osteoblast activity on the concave (or flexor) side of the tibiotarsal skeleton. These findings indicate that the bending deformity due to reduced mechanical integrity of the collagenous template is also associated with aberrant bone remodeling. (J Oral Sci 58, 255-263, 2016).