Feather buds exert a polarizing activity when transplanted to chick limb buds

Homeoproteins have been shown to be expressed in a position-specific manner along the anterior-posterior axis in the developing chick feather bud, as seen also in the developing limb bud. These facts raise the possibility that there may be common mechanistic features in the establishment of the anterior-posterior polarity between both organs. In order to investigate this possibility, feather bud tissues were transplanted into the anterior region of limb buds to determine whether feather bud tissues possess properties such as the zone of polarizing activity of the limb bud. The manipulated limb bud formed a mirror image duplication of the skeletal elements, mainly (2)2234 digit pattern or sometimes 3(2)234. Both the anterior and posterior halves of feather bud tissue exhibited almost equal activity in inducing ectopic skeletal elements. Hox d-12 and Hox a-13 were expressed coordinately around the transplanted site of the operated limb bud. This secondary axis-inducing activity of the feather bud was enhanced when grafts were pretreated with trypsin. In contrast, the presumptive feather bud tissue and inter-feather bud tissue did not induce a secondary axis of the limb bud. These results suggest that the feather bud contains a region that exerts polarizing activity and that this region may play key roles in the formation of the anterior-posterior and, if it exists, proximal-distal axis of the feather bud, possibly via the regulation of region specific expression of Hox genes.     

Temporal and spatial expression of a position specific antigen, AV-1, in chick limb buds

In a previous study, we demonstrated the presence of a position-specific antigen (AV-1) in chick limb buds at an early developmental stage. Here, we reported the temporal and spatial expressions and the biochemical characterization of the AV-1 antigen. Indirect immunofluorescence staining and immunoblot analysis clearly showed that the AV-1 antigen is a glycoprotein that is localized on the plasma membrane and that it is expressed from stage 19 and highly expressed at stages 22-26 in some middle-distal to anterior-distal region of limb buds. In the wing bud, at stage 28, the AV-1 antigen was faintly detected in the restricted space between the precartilaginous regions of the radius and the ulna, and those of the metacarpals 2 and 3, but not those of the metacarpals 3 and 4. Such stage-specific and "position-specific" expressions of the AV-1 antigen in limb buds strongly suggest that the AV-1 antigen or cells containing it are involved in determination of the limb pattern formation.     

Distribution of retinoids in the chick limb bud: analysis with monoclonal antibody

Retinoic acid induces anteroposterior duplicate formation in developing chick limb bud, and it may be a natural morphogen involved in limb pattern formation. Retinoic acid is produced from retinol locally in the limb bud via retinal, and thus, to elucidate the distribution of these retinoids in the limb bud seems to be important for the understanding of the morphogen formation. We produced a monoclonal antibody against the retinoids with BSA-RA (bovine serum albumin-retinoic acid) conjugate for antigen, and investigated the distribution of retinoids in the chick limb bud. The antibody predominantly bound to retinoic acid, but weakly to retinol and retinal. Retinoids appeared in the limb bud at stage 18 and were distributed through stages 20-24, when the pattern formation in distal mesoderm was in progress. Initially they were found evenly in the whole mesoderm, but disappeared gradually from core mesoderm and remained only in the region of peripheral mesoderm at stage 24. At stage 26, retinoids were detected only in ectoderm. These results support the idea that the retinoids actually play roles in limb pattern formation and suggest that the retinoids in the peripheral mesoderm are important for pattern formation. Further, the role of retinoids in epidermis development at later limb bud stages is also suggested.     

Congenic method in the chick limb buds by electroporation

Electroporation is a powerful tool with which to study limb development. Limb development, however, remains an intricate series of events, requiring the precise dissection of developmental processes using relevant transgenes. In this review, we describe the anatomy of the limb field as the basis of targeted electroporation, and specific expression vectors are discussed. We share a useful protocol for electroporation of chick limb buds, and the expression pattern of enhanced green fluorescent protein in the limb buds is used to demonstrate relevant embryonic patterning. Finally, useful trouble-shooting techniques are described.     

Chorio-allantoic membrane grafting of chick limb buds as a class practical

A method of carrying out grafts of early embryonic chick limb buds to the chick chorio-allantoic membrane is described which greatly reduces the time required for the procedure, demands a low level of manipulative skill, and does not require any complex media or equipment. A processing schedule which renders cartilage elements visible in whole mount and which can be completed in three hours, rather than the usual period of several days, is also described. Together, these improvements make it very much easier to use the technique as a class practical for large groups of students. Some implications that might be drawn from the results are indicated.     

Juxtaposition of two heterotypic monolayer cell cultures of chick limb buds

In chick limb buds, mesenchymal cells of the progress zone (PZ-cells) at different developmental stages segregate one from the other in mixed cell cultures, suggesting they have different cell affinity. In order to learn the possible roles of such differences in the cells, two heterotypic leg PZ-cell populations (cells from stages 25/26 and 20/21) in vitro were juxtaposed to allow them to form the boundary. A method with double cylindrical columns was used to make adjoining monolayer cell cultures. It was shown that heterotypic juxtaposition produced two chondrogenic patterns along the boundary: aggregates of chondrocytes formed by stage 20/21 PZ-cells and a chondrocyte-free band formed by those at stage 25/26. Juxtaposition of PZ-cells and proximal cells also formed these patterns, while that between cells from anterior and posterior PZ formed indistinct patterns along the boundary. Homotypic PZ-cell juxtaposition did not produce these patterns. The results suggest that different cell affinity has a role in the segmentation of cartilage patterns at a point along the proximodistal axis, as well as a role in retaining cells in one area so as not to be recruited to other condensation areas.     

Equilibrium binding parameters of an autoimmune monoclonal antibody specific for double-stranded DNA

Two techniques have been developed to estimate binding parameters for Jel 241 under equilibrium conditions. Jel 241 is an autoimmune monoclonal antibody derived from an NZB/NZW mouse which binds to double-stranded DNA. Thermal denaturation profiles of poly[d(AT)] were measured in the presence of increasing concentrations of IgG Jel 241. From these data it was estimated that the IgG occludes 12 base-pairs on duplex DNA, and the binding to double-stranded DNA was at least four orders of magnitude greater than to single-stranded DNA. In addition, intrinsic association constants (K(O)) were measured by a gel filtration technique for the interaction of both Fab and IgG Jel 241 to native calf thymus DNA. K(O) for the IgG was only 60-fold greater than for the Fab fragment for which K(O) was 4.4 X 10(4) M-1 at an NaCl concentration of 150 mM. Also, K(O) for the Fab increased dramatically with decreasing ionic strength, suggesting that there are four phosphates involved in the interaction. These techniques should be applicable to most autoimmune antibodies which bind to nucleic acid polymers.     

Double anterior chick limb buds and models for cartilage rudiment specification

Most models for the specification of the skeletal elements in the developing limb bud are based on a chemical specification well before overt cartilage differentiation. By contrast, a physico-mechanical model proposes that the process of condensation--an early feature of cartilage differentiation--is itself the basis for patterning the elements. The models thus make quite different predictions as to when the rudiments are specified. Double anterior limb buds have been constructed at stages earlier than condensation, with the expectation that, if specification of the humerus occurs before cartilage condensation, then limbs containing two humeri should develop, since the presumptive humerus lies largely in the anterior region. The development of anterior and posterior parts, on their own, was in general, consistent with the fate map; both developed a humerus that was thinner than normal. Double anterior limbs developed two humeri in 28% of cases and a much thicker humerus in 39%. These results strongly support models based on an early specification of limb rudiments and cannot be accounted for by the physical model. Double anterior limbs in which the two parts were from different stages, developed such that a digit 3 could lie adjacent to the radius, giving further striking evidence for early specification and local autonomy of development.     

Normal development of the skeleton in chick limb buds devoid of dorsal ectoderm

It has been suggested that the ectoderm on the dorsal and ventral faces of the limb bud plays a part in controlling the pattern of cartilage differentiation. To test this, the dorsal wing bud ectoderm in the chick embryo was destroyed by irradiation with ultraviolet light at stage 17-19, at the very beginning of limb bud development, but the apical ectodermal ridge was spared. The irradiated ectoderm disappeared within 24 hr (by stage 23-24) and did not regenerate thereafter; thus the dorsal surface of the limb bud was kept denuded throughout most of the period of skeletal pattern formation. By 6 or 7 days after the irradiation (stage 35), when the rudiments of all the adult skeletal elements are normally present in recognizable form, the irradiated wings could be placed into two categories, those that were approximately normal in shape and those that had curled dorsally. All of these limbs were reduced in size, to varying degrees, when compared to their controls and lacked dorsal soft tissues. The limbs that were normal in shape, however, even though sometimes denuded over practically the whole extent of their dorsal surface, almost always had a complete and normally proportioned cartilage pattern, suggesting that ectoderm (other than the apical ectodermal ridge) does not exert any direct control over the development of the limb cartilage pattern. However, many of those limbs that had curled as a result of the irradiation did have major pattern deformities, suggesting that the topology of cartilage differentiation does depend on the shape of the limb bud.     

The morphology and hormonal responsiveness of developing skeletal elements in chick limb buds

While parathyroid hormone (PTH), calcitonin (CT), and certain prostaglandins (PGs) are known to regulate the metabolism of both osteogenic and osteolytic cells of the adult skeleton through an adenosine 3', 5'-monophosphate-dependent mechanism, little is known about the development of this hormonally mediated response in embryonic skeletal tissues. In the present study, the responsiveness of embryonic skeletal elements to PTH and PGE2 was examined during various stages of development utilizing cAMP concentrations as an indicator of hormone-receptor interaction. The cytology of the limb skeletal system was examined also at each stage tested in order to compare the differentiated cellular phenotypes with their hormonal responsiveness. Prior to differentiation of cartilaginous elements in developing limb buds (stage 20-21), cells were responsive to PGE2 and epinephrine (EPI) but not to PTH. The first consistent response to PTH occurred coincident with the initial differentiation of the cartilage phenotype in limb buds (stage 24-25). A responsiveness to both PTH and PGE2 was progressively increased as maturation of cartilaginous and osteogenic elements occurred (stage 26-35). The initial response to CT was detected within cartilage rods in which osteogenic cells had differentiated (stage 33-35). The results of this study indicate that PGE2-sensitive cells exist within the developing limb prior to cytodifferentiation. The development of PTH responsiveness within embryonic chick limb buds is correlated with the onset of both chondrogenesis and osteogenesis in vivo.     

DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer

Monoclonal antibodies specific for the cyclobutane pyrimidine dimer (CPD) are widely used for detection and quantification of DNA photolesions. However, the mechanisms of antigen binding by anti-CPD antibodies are little understood. Here we report NMR analyses of antigen recognition by TDM-2, which is a mouse monoclonal antibody specific for the cis-syn-cyclobutane thymine dimer (T[c,s]T). ³¹P NMR and surface plasmon resonance data indicated that the epitope recognized by TDM-2 comprises hexadeoxynucleotides centered on the CPD. Chemical shift perturbations observed for TDM-2 Fab upon binding to d(T[c,s]T) and d(TAT[c,s]TAT) were examined in order to identify the binding sites for these antigen analogs. It was revealed that d(T[c,s]T) binds to the central part of the antibody-combining site, while the CPD-flanking nucleotides bind to the positively charged area of the V_H domain via electrostatic interactions. By applying a novel NMR method utilizing a pair of spin-labeled DNA analogs, the orientation of DNA with respect to the antigen-binding site was determined: CPD-containing oligonucleotides bind to TDM-2 in a crooked form, draping the 3′-side of the nucleotides onto the H1 and H3 segments, with the 5′-side on the H2 and L3 segments. These data provide valuable information for antibody engineering of TDM-2.     

Thermodynamics of oligosaccharide binding to a monoclonal antibody specific for a Salmonella O-antigen point to hydrophobic interactions in the binding site.

The thermodynamic characteristics of oligosaccharide binding to an antibody binding site that is dominated by aromatic amino acids suggest that the hydrophobic effect contributes substantially to complex formation as well as hydrogen bonding and van der Waals interactions. A detailed titration microcalorimetric study on the temperature dependence of the binding of a trisaccharide, representing the epitope of a Salmonella O-antigen, showed that its maximum binding to the monoclonal antibody Se155-4 occurs just below room temperature and both enthalpy and entropy changes are strongly dependent on temperature in a mutually compensating manner. The heat capacity change also shows an unusually strong temperature dependence being large and negative above room temperature and positive below. van't Hoff analysis of the temperature dependence of the binding constant yielded a biphasic curve with two apparent intrinsic enthalpy estimations (approximately -100 kJ mol-1 above 18 degrees C and approximately +100 kJ mol-1 below), each very different from the calorimetrically determined enthalpies (ranging from about -60 kJ mol-1 to -20 kJ mol-1). This was interpreted as being due to large enthalpy contributions from concomitant reactions, most notably changes in solvation. Linear plots, -delta H0 versus -T delta S0, observed for temperature-dependent measurements mirror the behavior seen for a series of functional group replacements, suggesting that the molecular and physical origin of these phenomena are closely related and linked to the role of water in complex formation. The thermodynamic results are compared to the mode of binding determined from a 2.05-A resolution structure of the Fab-oligosaccharide complex, and with literature data for the heat capacities of sugars in aqueous solution and for the thermodynamics of carbohydrate binding to transport proteins and lectins.     

Identification of a dihydropyridine-sensitive calcium channel in chick brain by a monoclonal antibody

A Balb/c mouse was immunized with chick synaptic plasma membranes and monoclonal antibodies were produced by fusion of spleen cells with NS-1 mouse myeloma cells. One antibody, MAC-L1, immunoprecipitated more than 90% of the [3H]PN200-110-labeled calcium channels but only 20% of the omega -conotoxin receptor solubilized from the chick brain membranes. Thus possibly, a certain portion of the omega -conotoxin receptor in the chick brain is a dihydropyridine-sensitive calcium channel. By the specific immunoprecipitation of 125I-labeled proteins, two large polypeptides of 193kDa and 130kDa under reducing conditions were identified as the major components of the calcium channel.     

Epithelial-mesenchymal interactions in the outgrowth of limb buds and facial primordia in chick embryos.

The facial primordia in the chick embryo begin as rounded swellings that surround the primitive mouth and these grow out to form the beak. The control of proximodistal outgrowth is not well understood but may involve similar mechanisms to the limb bud. In order to test this hypothesis, combinations were made between epithelium and mesenchyme from facial primordia and limb buds. Signals from all three types of facial mesenchyme (frontonasal mass, mandibular, and maxillary) maintained the thickened apical ectodermal ridge of limb epithelium for up to 48 h. Combinations of tissues from the frontonasal mass mesenchyme and limb epithelium underwent substantial and correct morphogenesis. In contrast, poor development was observed in combinations with mandibular mesenchyme. Signals from frontonasal mass epithelium promoted outgrowth and morphogenesis of limb mesenchyme whereas mandibular and maxillary epithelium did not support joint morphogenesis. The results suggest that signals employed in the epithelial-mesenchymal interactions in facial primordia are similar but not identical to those signals used in the limb bud.     

Role of chondrogenic tissue in programmed cell death and BMP expression in chick limb buds

In the developing chick leg bud, massive programmed cell death occurs in the interdigital region. Previously, we reported the inhibition of cell death by separation of the interdigital region from neighboring digit cartilage. In this study, we examined the relationship between cell death and cartilaginous tissue in vitro. First, cell fate was observed with DiI that was used to examine cell movement in the distal tip of leg bud. Labeled cells in the prospective digital region were distributed only in the distal region as a narrow band, while cells in the prospective interdigital region expanded widely in the interdigit. In coculture of monolayer cells and a cell pellet tending to differentiate into cartilage, monolayer cells migrated into the cell pellet. These results suggested that digit cartilage tends to recruit neighboring cells into the cartilage during limb development. Next, we observed the relationship between cell death and chondrogenesis in monolayer culture. Apoptotic cell death that could be detected by TUNEL occurred in regions between cartilaginous nodules in mesenchymal cell culture. More apoptotic cell death was detected in the cell culture of leg bud mesenchyme of stage 25/26 than that of leg bud mesenchyme of stage 22 or that of stage 28. The most developed cartilaginous nodules were observed in the cell culture of stage 25/26. Finally, we observed Bmp expression in vitro and in vivo. Bmp-2, Bmp-4 and Bmp-7 were detected around the cartilage nodules. When the interdigit was separated from neighboring digit cartilage, Bmp-4 expression disappeared near the cut region but remained near the digit cartilage. This correlation between cell death and cartilaginous region suggests that cartilage tissue can induce apoptotic cell death in the developing chick limb bud due to cell migration accompanying chondrogenesis and Bmp expression.