On the migration of epidermal melanoblasts in the avian embryonic wing bud

Summary After heterotopic grafting of quail neural crest cells to the wing buds of embryos of an unpigmented chicken strain, epidermal melanocytes of donor origin are found almost exclusively distal from the graft in the host's epidermis. This directed cell migration ceases, if the apical ectodermal ridge (together with a small amount of subridge mesoderm) is removed from the operated wing buds or if impermeable materials are interposed between it and the rest of the wing bud. Under these conditions epidermal melanocytes are found not only distal from but also proximal to the grafts. From this it may be deduced that the apical ectodermal ridge directs the migration of epidermal melanoblasts in the avian embryonic wing bud, possibly by a chemotactic mechanism. The presence or absence of the apical ectodermal ridge had no observable effect on the migratory behaviour of other neural crest derived cell populations (Schwann cells and non-epidermal melanocytes) in the wing bud. This shows that the apical ectodermal ridge specifically influences epidermal melanocytes.This work was supported by the Österreichischer Fonds zur Förderung der wissenschaftlichen Forschung (P 4680)     

Hyaluronic acid influences the migration of myoblasts within the avian embryonic wing bud

Myoblasts migrate in a proximodistal direction within the avian embryonic wing bud during normal limb development. Since the presence and distribution of hyaluronic acid within the wing bud coincide with the time and with the direction of the migration of myoblasts, we microinjected hyaluronic acid into chicken wing buds that had received grafts containing quail myoblasts. It was found that injected hyaluronic acid has a strong positive effect on the migration of myoblasts: it causes a migration of myoblasts in donor-host combinations in which this is normally not the case, and it can cause migration in a proximal direction, a phenomenon not observed during normal development. From this it may be concluded that hyaluronic acid can influence myoblast migration in vivo. A similar effect could be observed after the microinjection of dextran sulfate, a synthetic compound having similar physicochemical properties. Hyaluronic acid, therefore, may play an important role in the control of the migration of myogenic cells in vivo by its physicochemical properties.     

The orientation of the wing mesenchyme influences the direction of the migration of myoblasts in the avian embryonic wing bud

Summary In order to analyze the influence of the orientation of the wing bud mesenchyme on the proximodistal direction of the migration of myoblasts in the avian embryonic wing bud, blocks of wing-bud mesenchyme were cut out and rotated around a dorso-palmar axis through 90° or 180°. Tissues originating from the quail wing bud and containing myoblasts were grafted into the space between the wing mesenchyme and the rotated blocks of mesenchyme proximal to the latter. In all experiments the donor-embryos were older than the acceptor-embryos. From HH stage 24 on, the rotation of the block of mesenchyme inhibited the migration of the myoblasts in a distal direction. We therefore propose that the orientation of the wing bud mesenchyme has an influence on the migratory behavior of myoblasts. This influence could provide “directional information” for the migrating myoblasts, allowing the migration of myoblasts in a distal direction only.     

The formation of premuscle masses during chick wing bud development

Summary The skeletal musculature of chick limb buds is derived from somitic cells that migrate into the somatopleure of the future limb regions. These cells become organized into the earliest muscle primordia, the dorsal and ventral premuscle masses, prior to myogenic differentiation. Therefore, skeletal-muscle specific markers cannot be used to observe myogenic cells during the process of premuscle mass formation. In this study, an alternative marking method was used to determine the specific stages during which this process occurs. Quail somite strips were fluorescently labeled and implanted into chick hosts. Paraffin sections of the resulting chimeric wing buds were stained with the monoclonal antibody QH1 in order to identify graft-derived endothelium. Non-endothelial graft-derived cells present in the wing mesenchyme were assumed to be myogenic. At Hamburger and Hamilton stage 20, myogenic cells were distributed throughout the central region of the limb, including the future dorsal and ventral premuscle mass regions and the prechondrogenic core region. By stage 21, the myogenic cells were present at greater density in dorsal and ventral regions than in the core. By stage 23, nearly all myogenic cells were located in the dorsal and ventral premuscle masses. Therefore, the two premuscle masses become established by stage 21 and premuscle mass formation is not complete until stage 23 or later. Premuscle mass formation occurs concurrently with early chondrogenic events, as observed with the marker peanut agglutinin. To facilitate the investigation of possible underlying mechanisms of premuscle mass formation, the micromass culture system was evaluated, to determine whether or not it can serve as an accurate in vitro model system. The initially randomly distributed myogenic cells were observed to segregate from prechondrogenic regions prior to myogenic differentiation. This is similar to myogenic patterning in vivo.     

Mathematical modelling of the growth processes in the developing chick wing bud

This paper illustrates how a simple geometric model resembling the shape of the chick wing bud at an early growth stage can be mathematically expanded to simulate subsequent growth characteristics of the developing bud. The model was tested against several sets of experimental data and gave an acceptable representation of growth over the range considered. Representing growth patterns in this form enables the determination of differential growth characteristics in different parts of the bud and provides boundary constraints which will play an important part in the eventual evaluation of internal growth mechanisms.     

Endothelial heterogeneity in the chick wing bud: A morphometric study

Summary The microvascular endothelium of the chick wing bud at stages 22, 27, and 32 was evaluated by ultrastructural morphometry. The rationale for this study is based on the hypothesis that endothelial cells exhibit variation in structure and function during cytodifferentiation. The microvessels had a luminal diameter range such that they were classified as capillaries. The thin continuous endothelium was devoid of a basal lamina. The endothelium had a very small number of plasmalemmal vesicles; vacuoles were however present for all stages and in some cases were abundant. The temporal findings were that endothelial cell thickness increases, plasmalemmal vesicle densities decrease, and the densities of cytoplasmic vacuoles increase. The spatial results were that endothelial cells in proximal regions of the limb have a greater thickness, contain fewer vesicles and have more vacuoles than those in distal regions. In general, these results indicate that endothelial ultrastructural heterogeneity occurs within a 31/2 day timespan of wing bud development. The discussion considers the results with regard to recent reports on endothelial cell heterogeneity.     

The lymphatic endothelium of the avian wing is of somitic origin.

It has recently been shown that there are lymphangioblasts in the early avian wing bud, but fate map studies on the origin of these cells have not yet been performed. The lymphatics in the wings of 10-day-old chick and quail embryos are characterized by both the position along with all major blood vascular routes and by the Vascular Endothelial Growth Factor Receptor-3 (VEGFR-3) expression. In the quail, the endothelium of both blood vessels and lymphatics can be marked with the QH1 antibody. We have grafted the dorsal halves of epithelial somites of 2-day-old quail embryos homotopically into chick embryos. The grafting was performed at the wing level and the host embryos were reincubated until day 10. The chimeric wings were studied with the QH1 antibody alone and with double staining consisting of VEGFR-3 in situ hybridization and QH1 immunofluorescence. Our results show that in the wing the endothelium of both the blood vessels and the lymphatics is derived from the somites. QH1-positive endothelial cells form the vasculature of the chimeric wings. Chimeric lymphatics of the wing can be identified because of their typical position and their VEGFR-3 and QH1 double-positivity. This shows that not only the blood vascular cells but also the lymphatic endothelial cells of the avian wing are born in the paraxial/somitic mesoderm.     

Molecular heterogeneity of chondroitin sulphate in the early developing chick wing bud

Proteoglycans are ubiquitous extracellular matrix molecules whose role in development remains poorly understood. In the developing chick limb, the nature and possible roles of a number of extracellular matrix proteins is well documented. Much less is known of the biochemical nature, and more importantly, the roles of proteoglycans. Using a panel of monoclonal antibodies (Mabs) which recognise specifie epitopes on the constituent chondroitin/dermatan sulphate chains, we show that distinct sub-populations of proteoglycans are dynamically expressed within the limb ectoderm, the ectodermal basement membrane and the limb mesenchyme. In particular, prior to chondrogensis, chondroitin-6-sulphate-rich proteoglycans containing over-sulphated domains reside predominantly within the mesenchymal extracellular matrix ECM, whilst chondroitin-4-sulphate (C-4-S) is associated with the ectodermal basement membrane and subjacent mesenchymal ECM. At stage 24, C-4-S is also localized in the prechondrogenic condensation. Concomitantly with overt chondrogenesis, the epitopes recognized by the Mabs become restricted to the chondrifying skeletal elements and the undifferentiated distal mesenchyme. The significance of these findings has yet to be elucidated.     

Tubedown-1, a novel acetyltransferase associated with blood vessel development.

We have used an embryonic endothelial cell line (IEM cells) as an experimental system for identifying and characterizing new molecules which are regulated during blood vessel development. A novel gene isolated from IEM cells, tubedown-1 (tbdn-1), is expressed at high levels in unstimulated IEM cells and is downregulated during formation of capillary tube structures by the IEM cells induced by basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) in vitro. Tbdn-1 is also downregulated in M1 myeloid leukemia cells after differentiation in response to LIF in vitro. Tbdn-1 is homologous to the yeast NAT-1 N-terminal acetyltransferases and encodes a novel protein of approximately 69 kDa associated with an acetyltransferase activity. Levels and distribution of tbdn-1 expression are regulated in both endothelial and hematopoietic cells during development in tissues such as the yolk sac blood islands, heart, and liver blood vessels. In the adult, tbdn-1 expression is low or undetected in most organs examined with the exception of the atrial endocardium, the endothelial and myeloid compartments of bone marrow, and the remodeling vascular bed of atretic ovarian follicles. The distribution and regulation of expression of tbdn-1 suggest that this novel acetyltransferase may be involved in regulating vascular and hematopoietic development and physiologic angiogenesis.     

Taste bud development in the zebrafish, Danio rerio.

Taste buds are chemosensory endorgans consisting of modified epithelial cells. Fish and other vertebrates use their taste bud cells to sample potential food, either selecting or rejecting substances according to their edibility. The adult gustatory system in fish has been studied thoroughly, including regeneration experiments. Taste buds occur in the epithelia of the lips, the mouth cavity, the oropharyngeal cavity, and also in the skin of the barbels, the head, and sometimes even all over the body surface. Despite its importance for feeding, little is known about the ontogeny of the fish taste system. We examined the development of taste buds in the zebrafish on the light microscopical and the scanning and transmission electron microscopical levels. Taste buds develop later than the olfactory organ and the solitary chemosensory cells, two other chemosensory systems in aquatic vertebrates. The first few taste bud primordia are visible within the epithelia of lips and gill arches 3 to 4 days after fertilization, and the first few taste buds with open receptor areas appear on the lips and simultaneously on the gill arches 4-5 days after fertilization, which coincides with the onset of feeding. Taste buds in the mouth cavity, on the head, and on the barbels are formed later in development. As seen in other fish, zebrafish taste buds contain elongate dark and light cells, termed according to their electron density. Dark cells with a cell apex of many short microvilli appear first, followed by the light cells with one large microvillus. In addition, the zebrafish has a third fusiform cell type, which appears last. This cell type is low in electron density and has a brush-like apical ending with several small microvilli. This cell type has not been described previously. Furthermore, in zebrafish, the ontogenetic processes of taste bud formation differ from regenerative processes described in the literature.     

The control of directed myogenic cell migration in the avian limb bud

Summary Interspecific grafting experiments between chick and quail embryos were carried out in order to investigate the mechanism controlling myogenic cell migration in the avian limb bud. In six series, various experimental set-ups were prepared involving different age combinations of donor and host. The migration of the myogenic cells contained nor and host. The migration of the myogenic cells contained in the quail donor could be traced due to the prominent perinucleolar heterochromatin of the quail nucleus. Irrespectively of the presence or absence of the apical ectodermal ridge (AER), myogenic cells were found to migrate distally when implanted at a more distal site or into a younger host. They were even found to migrate in the reverse direction when younger host tissue was located proximal to the graft.From these findings, we conclude that the state of differentiation (juvenility) of the limb bud mesenchyme controls the directed migration of myogenic cells.     

Participation of individual brachial somites in skeletal muscles of the avian distal wing

In this paper we investigate the somitic origin of the individual muscles of the forearm and hand using quail-chick chimeras. Our results show that only somites 16–21 give rise to wing muscle, but they take part in muscle formation to different extents. Somite 21 does not always participate in the formation of muscle of the forearm and hand. The most cranial somite (16) takes part in the radial muscles and the most caudal somites (20, 21) in the ulnar muscles, reflecting their position with respect to the limb bud. The centrally located somites (17, 18, 19) are involved in all (18) or most (17, 19) muscle primordia. This pattern of distribution is clearest in the forearm, whereas the participation of somites in particular muscle groups is not so distinct in the hand. Hand muscles are mainly made up of cells from somites 18–20. All brachial somites participate in dorsal (extensor) as well as ventral (flexor) muscles of the forearm and hand. Each somite takes part in more than three muscle primordia in a reproducible fashion, and every muscle primordium is derived from at least three somites. Especially the M. ulnimetacarpalis ventralis takes origin from all somites involved in limb muscle formation (16–21). Apart from muscle cells, endothelial cells also and a few fibroblasts of quail origin are found in the limb bud after somite grafting.     

Region-specific expression of mario reveals pivotal function of the anterior nondigit region on digit formation in chick wing bud.

We report the region-specific expression of a novel gene, named mario, whose expression domain is in the distal tip of the presumptive and developing digit 2 region in the developing chick wing bud. The anterior region-specific expression of mario corresponds well with the presence of digit 2, and fate map analysis showed that mario expression at early stages represents the presumptive digit 2 region. Using mario expression as a region-specific marker for the digit 2 region, several surgical operations were performed to obtain insights into digit 2 development in the chick wing. Cell fate tracing concomitant with a zone of polarizing activity (ZPA) implantation revealed that an additional digit 2 in the ZPA implantation into the anterior or middle region of wing bud is derived from the original digit 2 region (mario-positive region). Surgical manipulations revealed that the anterior nondigit region has an inhibitory effect on digit 2 formation. Taken together, these results suggest that the most-anterior region, including the anterior necrotic zone, restricts the position of digit 2 region by limiting the anterior border of the digit 2 region and preventing its expansion.     

The distribution of mesenchyme proteoglycan (PG-M) during wing bud outgrowth

Summary This study utilizes immunofluorescence to describe the distribution of several extracellular matrix molecules in the chick embryo during the process of limb outgrowth and the formation of precartilage condensations. A large chondroitin sulfate proteoglycan (PG-M) is detected at the wing level at Hamburger and Hamilton stage 14 in and under the dorsal ectoderm, and is associated with the basement membranes around the neural tube, notochord and pronephros, but not with other basement membranes. The galactose-specific leetin, peanut agglutinin (PNA), has a similar distribution except that it also binds to the dorsal side of the neural tube. PG-M is not detected in the limb mesenchyme until after stage 17, when it is present in the distal region, as is PNA-binding material. With further development of the wing bud, PG-M is present in the subectodermal mesenchyme, the mesenchyme at the distal tip and in the prechondrogenic core. After stage 22 PNA-binding material becomes localized in the prechondrogenic core, the basement membranes under the apical ectodermal ridge, and the ventral sulcus. The distribution of these components (PG-M and PNA binding material) overlaps, but differs from that of type I collagen and fibronectin and basement membrane components, such as laminin, basement membrane heparan sulfate proteoglycan, and type IV collagen. Tenascin, on the other hand, is not detected in the limb bud until stage 25, after the appearance of cartilage matrix components such as type II collagen and cartilage proteoglycan (PG-H). These results are considered in relation to the formation of precartilage aggregates, and indicate that PNA binds to components in precartilage aggregates other than PG-M or tenascin.     

A study on skeletal myogenic cell movement in the developing avian limb bud

Summary Quail limb mesenchyme containing myogenic cells of somitic origin were transplanted into chick limb buds to determine whether cell movement might play a role in avian limb myogenesis. In general, cell displacement was not detected 1-day after implantation: all quail cells were found at the graft site. Migration was evident 2-days after implantation but not all cell types were capable of movement; myogenic cells were very invasive while chondrocytes were relatively immobile. The spreading of myogenic cells was discernible up to 4-days after implantation and specifically in a proximodistal direction towards the apex of the limb.