Presumptive Dermis Research Articles

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 Hurle's 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.

Development of Collagen Fibres and Lysyl Oxidase Expression in the Presumptive Dermis of Chick Limb Bud

Lysyl oxidase (LOX) plays a critical role in the formation of cross-linkages in extracellular matrix molecules. Thus, it is essential for the biogenesis and homeostasis of the connective tissue matrix. During development, collagen fibres and elastic system fibres emerge and accumulate in a temporospatial manner in the presumptive dermis of chicks. In this study, we investigated LOX mRNA expression by laser capture microdissection and RT-qPCR and LOX protein localization by immunohistochemistry. The picrosirius polarization method was used to investigate a relation between collagen accumulation and LOX expression. PCR analysis showed that the expression of LOX mRNA in the presumptive dermis became apparent at embryonic day 13 and increased considerably by ED17. Immunohistochemical staining for LOX in the dermis was very low at all stages of development. Accumulation of collagen fibres was seen in the dermis on ED10, and higher wavelengths of birefringence became evident by ED13. Our findings suggest that the temporal pattern of LOX mRNA expression correlates with collagen fibre accumulation in the dermis of the developing chick limb bud, whereas LOX expression was relatively constant at the protein level.

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.

Subectodermal microfibrillar bundles are organized into a distinct parallel array in the developing chick limb bud

In this study, a unique fiber system in the subectodermal mesenchyme of the chick limb bud was visualized immunohistochemically with the use of a novel monoclonal antibody termed "FB1." This antibody stained a subset of extracellular fibers in the embryonic mesenchyme. Among the fibers visualized, those running perpendicularly to the limb bud ectoderm became progressively prominent in their thickness and length, and organized into a parallel array in the subectodermal region. This fiber system was distinct from that of major collagens, fibronectin, or tenascin. A molecule immunoprecipitated with FB1 comigrated with JB3 antigen, or chicken fibrillin-2. The fibers visualized immunohistochemically by FB1 and JB3 were indistinguishable from each other, and ultrastructurally appeared to be bundles composed of tubular-like microfibrils that originated directly from the ectodermal basal lamina. They lacked the amorphous deposits that are characteristic of elastin. A similar array of subectodermal fibers was also found in the developing axilla and some truncal regions, again well before the development of a definitive dermis. These findings suggest that a parallel array of subectodermal FB1-positive fibers constitutes a precocious fiber system in the presumptive dermis prior to the substantial formation of collagenous fibers. These fibers could be developmentally linked to oxytalan fibers, which are known to be present in the papillary dermis in mature cutaneous tissue.

Changing patterns of cell adhesion molecules during mouse pelage hair follicle development. 1. Follicle morphogenesis in wild-type mice.

The morphogenesis of hairs is initiated and maintained by reciprocal interactions between groups of epithelial and mesenchymal cells. To examine whether cell adhesion molecules play a role in this process, prenatal distribution patterns of various cell adhesion molecules were studied during hair follicle morphogenesis in the dorsal skin of C57BL mouse embryos, using monoclonal antibodies. E-cadherin was present on all epithelial cells of the skin when the ectoderm gave rise to periderm and epidermis. E-cadherin was reduced in the follicle placodes and hair plugs, then disappeared from the presumptive hair matrix of elongating follicles. P-cadherin was initially present on all cells of periderm and epidermis and was later retained at a reduced level in the basal epidermal layer. P-cadherin was prominent in all follicle placodes and hair plugs and in the presumptive hair matrix of elongating follicles. N-CAM was present on all mesenchymal cells of the presumptive dermis at the prefollicle stage, then temporarily restricted to a few cells just below the dermal-epithelial junction. Later, N-CAM reappeared in the interfollicular mesenchyme and was prominent in the mesenchymal sheath and dermal papilla of elongating follicles. In addition, N-CAM was expressed in the hair plugs, then became progressively restricted to the upper caudal part of the elongating follicles. The results suggest that the main role of cell adhesion molecules is to mould the follicle by relaxing or reinforcing cell contacts in areas of increased morphogenetic activity.

NCAM immunoreactivity during major developmental events in the rat maxillary nerve-whisker system

The distribution of immunoreactivity for neural cell adhesion molecule (NCAM) has been characterized during the formation of the trigeminal ganglion and during the process of axon outgrowth and target differentiation in the maxillary nerve-whisker system, in rat fetuses of known gestational age. Proliferating cells within the trigeminal placode are NCAM immunoreactive when first observed on embryonic day (E) 10. NCAM immunoreactivity is lost from placode-derived cells as they migrate to the trigeminal ganglion. It re-appears on ganglion cell somata and on centrally and peripherally projecting axons at the time of neurite outgrowth. NCAM-immunoreactive centrally projecting axons reach the developing brain stem two days before peripheral axons encounter the presumptive whisker pad. NCAM immunoreactivity on axons and somata is down regulated after P0, following target contact and whisker follicle differentiation. The presumptive dermis of the whisker pad at E13 appears as a sheet-like condensation of intensely NCAM immunostained cells. Discrete infraorbital row nerves can be identified on E13. These form in the subdermal region which contains only low levels of NCAM immunoreactivity. Condensations of NCAM immunostained mesenchyme replace the dermal sheet on E14 and each condensation is associated with a plexus of infraorbital nerve fibers. The epithelium overlying each condensation grows downward on E15. Focal epithelial regions become NCAM immunoreactive by E18. NCAM immunostaining within epithelial components of the whisker follicle is temporally correlated with contact by NCAM-immunoreactive infraorbital nerve fibers. The site restricted expression of NCAM immunoreactivity during trigeminal embryogenesis is consistent with the idea that NCAM plays an integral role in critical aspects of pattern formation in the maxillary nerve-whisker system, particularly in the organization of placode and non-placode derived trigeminal neuroblasts, axon outgrowth and in the differentiation of the vibrissae follicles.

Development of human embryonic and fetal dermal vasculature.

This report summarizes recent advances in the understanding of the structure and organization of the microvasculature in developing human skin. Previous observations suggested that the skin contains no blood vessels as late as eight weeks estimated gestational age (EGA). Computer reconstructions, in conjunction with light and transmission electron microscopy (TEM), however, demonstrated that specimens as young as 35-45 d show a level of vascular complexity previously unknown. The computer reconstructions showed that the vasculature was organized in one or two planes parallel to the epidermis. A simple, single plane was evident in specimens 40-50 d EGA, whereas specimens 50-75 d EGA showed two planes. Fewer vessels were continuous throughout the tissue sample in the younger specimens compared with the older specimens. Superior views highlighted the continuities and connections of vessels. The younger specimens showed more discontinuous segments of vessels when compared with the network established in the older specimens. In the earliest specimens examined morphologically (35-40 d), simple, capillarylike vessels were morphologically identifiable in presumptive dermis. The samples studied by TEM revealed detailed structure of the vessel wall including extreme attenuations and projections, plasmalemmal vesicles, and junctions similar to adult endothelial cells. Little or no basal lamina surrounded the vessel. The basal lamina first appeared in the form of amorphous deposits and eventually thickened and became continuous. By the end of the first trimester, the basal lamina still lacked the organization of adult cutaneous arterial and venous segments. These findings suggest that the major vascular organization of the dermis is defined in the first trimester of development.

Development of Human Embryonic and Fetal Dermal Vasculature

This report summarizes recent advances in the understanding of the structure and organization of the microvasculature in developing human skin. Previous observations suggested that the skin contains no blood vessels as late as eight weeks estimated gestational age (EGA). Computer reconstructions, in conjunction with light and transmission electron microscopy (TEM), however, demonstrated that specimens as young as 35-45 d show a level of vascular complexity previously unknown. The computer reconstructions showed that the vasculature was organized in one or two planes parallel to the epidermis. A simple, single plane was evident in specimens 40-50 d EGA, whereas specimens 50-75 d EGA showed two planes. Fewer vessels were continuous throughout the tissue sample in the younger specimens compared with the older specimens. Superior views highlighted the continuities and connections of vessels. The younger specimens showed more discontinuous segments of vessels when compared with the network established in the older specimens. In the earliest specimens examined morphologically (35-40 d), simple, capillarylike vessels were morphologically identifiable in presumptive dermis. The samples studied by TEM revealed detailed structure of the vessel wall including extreme attenuations and projections, plasmalemmal vesicles, and junctions similar to adult endothelial cells. Little or no basal lamina surrounded the vessel. The basal lamina first appeared in the form of amorphous deposits and eventually thickened and became continuous. By the end of the first trimester, the basal lamina still lacked the organization of adult cutaneous arterial and venous segments. These findings suggest that the major vascular organization of the dermis is defined in the first trimester of development.

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence: I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo

The aim of this work was to prepare specific antibodies against skin and bone collagen (type I) and cartilage collagen (type II) for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Antibodies against native type I collagen from chick cranial bone, and native pepsin-extracted type II collagen from chick sternal cartilage were raised in rabbits, rats, and guinea pigs. The antibodies, purified by cross-absorption on the heterologous collagen type, followed by absorption and elution from the homologous collagen type, were specific according to passive hemagglutination tests and indirect immunofluorescence staining of chick bone and cartilage tissues. Antibodies specific to type I collagen labeled bone trabeculae from tibia and perichondrium from sternal cartilage. Antibodies specific to type II collagen stained chondrocytes of sternal and epiphyseal cartilage, whereas fluorescence with intercellular cartilage collagen was obtained only after treatment with hyaluronidase. Applying type II collagen antibodies to sections of chick embryos, the earliest cartilage collagen found was in the notochord, at stage 15, followed by vertebral collagen secreted by sclerotome cells adjacent to the notochord from stage 25 onwards. Type I collagen was found in the dermatomal myotomal plate and presumptive dermis at stage 17, in limb mesenchyme at stage 24, and in the perichondrium of tibiae at stage 31.

Studies of skin of fetus and the child. The distribution of alkaline phosphatase in the skin of the fetus.

Alkaline phosphatase (AP) appears early in fetal life in the mesenchymal cells of the presumptive dermis, especially in the anlagen of the papillae of hair follicles and in the endothelial cells of the blood vessels. It appears later in the eccrine and apocrine sweat glands from which it later mostly disappears. AP is inconstantly present in the cells of the bulb of hair follicles, and it is never present in the cells of the sebaceous glands and the epidermis. In the tips of the fingers and toes it is concentrated in the nail wall, around the nail matrix and nail plate, and in the sole plate. Towards the end of fetal life, AP is only moderately active or absent in the tips of the fingers and toes. The physiological role of AP in the skin may be related to morphogenesis and particularly to the differentiation of the pilary system, and to keratinization of the epidermis and the nail plate. In the endothelium of small vessels its function could be that of aiding the transfer of materials to and from the vessels (phosphorylation and dephosphorylation). AP disappears from the endothelium of vessels as soon as the tunicae are formed; it remains in the arterial limbs of capillaries.