Apical ectodermal ridge induction by the transplantation of En-1-overexpressing ectoderm in chick limb bud

In the early chick embryo, the dorsal–ventral (DV) boundary organizes the apical ectodermal ridge (AER) structure in the limb bud field. Here it is reported that Engrailed-1 ( En-1 ), a homolog of the Drosophila segment polarity gene engrailed expressed in the ventral limb ectoderm, participates in AER formation at the DV boundary of the limb bud. Restricted ectopic expression of En-1 in the dorsal side of the limb bud by transplantation of En-1 -overexpressing ectoderm induces ectopic AER at the boundary of En-1 -positive and -negative cells. The results suggest that En-1 is involved in AER formation at the DV boundary of the limb bud.     

Effects of digit tissue on cell death and ectopic cartilage formation in the interdigital zone in chick leg buds

In developing chick leg buds, large-scale cell death occurs in the interdigital zone, which is responsible for the separation of digits from each other. Ectopic cartilage formation is known to occur upon removal of the chondrogenic digit tissue of the leg bud. To examine the mechanisms of ectopic cartilage formation in the interdigital cell death region, we performed the following operations on stage 28–29 leg buds: (i) removal of the digit-forming area; (ii) incision between the interdigital zone and digit region; (iii) insertion of an aluminum barrier into the interdigital zone; and (iv) insertion of a permeable Nuclepore filter into the interdigital zone. In all cases, the inhibition of cell death and/or the formation of ectopic cartilage in the interdigital zone were observed, although the frequency of the inhibition of cell death and the formation of ectopic cartilage varied, depending upon the position where the operations were performed. These results suggest that cell death and cell differentiation in the interdigital zone may be controlled by some factor(s) from digit cartilage.     

Functional analysis of chick heparan sulfate 6‐O‐sulfotransferases in limb bud development

Heparan sulfate (HS) interacts with numerous growth factors, morphogens, receptors, and extracellular matrix proteins. Disruption of HS synthetic enzymes causes perturbation of growth factor signaling and malformation in vertebrate and invertebrate development. Our previous studies show that the O‐sulfation patterns of HS are essential for the specific binding of growth factors to HS chains, and that depletion of O‐sulfotransferases results in remarkable developmental defects in Drosophila, zebrafish, chick, and mouse. Here, we show that inhibition of chick HS‐6‐O‐sulfotransferases (HS6ST‐1 and HS6ST‐2) in the prospective limb region by RNA interference (RNAi) resulted in the truncation of limb buds and reduced Fgf‐8 and Fgf‐10 expressions in the apical ectodermal ridge and in the underlying mesenchyme, respectively. HS6ST‐2 RNAi resulted in a higher frequency of limb truncation and a more marked change in both Fgf‐8 and Fgf‐10 expressions than that achieved with HS6ST‐1 RNAi. HS6ST‐1 RNAi and HS6ST‐2 RNAi caused a significant but distinct reduction in the levels of different 6‐O‐sulfation in HS, possibly as a result of their different substrate specificities. Our data support a model where proper levels and patterns of 6‐O‐sulfation of HS play essential roles in chick limb bud development.     

Apoptosis in the chick wing bud and the permanence of FGF-2 rescue

Summary Two regions of programmed cell death that occur in the mesoderm of developing chick wing buds were studied in vitro. The opaque patch (OP) and posterior necrotic zone (PNZ) were examined for the presence of internucleosomal DNA degradation and for rescue by protein synthesis inhibition, two defining characteristics of apoptosis. Agarose gel electrophoresis showed that DNA from OP and PNZ tissue was cleaved into nucleosome size pieces and this cleavage was prevented by inhibition of protein synthesis with cycloheximide. Both regions showed rescue with cycloheximide as determined by the chromium release assay and examination of electron micrographs. Also, the permanence of basic fibroblast growth factor (FGF-2) rescue in the OP and PNZ was examined using the chromium release assay. While rescue in the OP was found to be permanent, rescue in the PNZ only delayed death while FGF-2 was present in the culture medium. This research shows that death in the OP and PNZ exhibits internucleosomal DNA fragmentation and is prevented by inhibition of protein synthesis with cycloheximide, biochemically characterizing this death as apoptosis. It also suggests that in vitro FGF-2 rescue is permanent in the OP but is merely a delay of cell death in the PNZ.     

NuSerum,a synthetic serum replacement,supports chondrogenesis of embryonic chick limb bud mesenchymal cells in micromass culture

Summary In an effort to establish a more chemically defined culture system to study the regulation of chondrogenic differentiation in vitro, two commercially available serum replacements, NuSerum and NuSerum IV, were tested on embryonic limb mesenchyme. Limb bud (LB) mesenchymal cells were isolated from Hamilton-Hamburger stage 23–24 chick embryos and plated at various densities (1, 5, 10, or 20 × 10⁶ cells/ml) in micromass culture for 4 days in media supplemented with 10% fetal bovine serum (FBS), NuSerum or NuSerum IV. Cell growth was assessed by the incorporation of [³H]leucine and [³H]thymidine. Chondrogenesis was determined by the incorporation of [³⁵S]sulfate and by the number of Alcian blue-staining cartilage nodules. In high density (20 × 10⁶ cells/ml) cultures, which favored chondrogenic differentiation, both serum replacements supported protein synthesis and chondrogenesis equally well as FBS. In cultures plated at 5 × 10⁶ cells/ml, a cell density in which was chondrogenesis-limiting, both NuSerum and NuSerum IV significantly enhanced incorporation of [³⁵S]sulfate (2.6-fold), [³H]leucine (1.4-fold), and [³H]thymidine (1.9-fold), compared to FBS. Enhancement of chondrogenesis was also apparent by the increases in the number of Alcian blue-staining cartilage nodules and the ratio of sulfate: leucine incorporation in cultures plated at 5 × 10⁶ cells/ml. Interestingly, the localization of cartilage nodules was extended out to the periphery of micromass cultures fed with NuSerum or NuSerum IV. The observed effects of NuSerum and NuSerum IV may be attributed to a combination of factors, including lower concentrations of serum and its associated proteins, as well as supplemented growth factors and hormones known to promote cell proliferation and differentiation. Therefore, NuSerum and NuSerum IV are excellent, low-cost replacements for FBS in maintaining cellular growth and promoting chondrogenesis in LB mesenchymal cell cultures in vitro.     

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.     

Changes in matrix protein gene expression associated with mineralization in the differentiating chick limb-bud micromass culture system

Chick limb‐bud mesenchymal stem cells plated in high density culture in the presence of 4 mM inorganic phosphate and vitamin C differentiate and form a mineralizable matrix, resembling that of the chick growth plate. To further elucidate the mechanism that allows these cultures to form physiologic hydroxyapatite deposits, and how the process can be manipulated to gain insight into mineralization mechanisms, we compared gene expression in mineralizing (with 4 mM inorganic phosphate) and non‐mineralizing cultures (containing only 1 mM inorganic phosphate) at the start of mineralization (day 11) and after mineralization reached a plateau (day 17) using a chick specific microarray. Based on replicate microarray experiments and K‐cluster analysis, several genes associated with the mineralization process were identified, and their expression patterns confirmed throughout the culture period by quantitative RT‐PCR. The functions of bone morphogenetic protein 1, BMP1, dentin matrix protein 1, DMP1, the sodium phosphate co‐transporter, NaPi IIb, matrix metalloprotease 13. MMP‐13, and alkaline phosphatase, along with matrix protein genes (type X collagen, bone sialoprotein, and osteopontin) usually associated with initiation of mineralization are discussed. J. Cell. Biochem. 112: 607–613, 2011. © 2010 Wiley‐Liss, Inc.     

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.     

Involvement of Wnt-5a in chondrogenic pattern formation in the chick limb bud

Members of the Wnt family are known to play diverse roles in the organogenesis of vertebrates. The full-coding sequences of chicken Wnt-5a were identified and the role it plays in limb development was examined by comparing its expression pattern with that of two other Wnt members, Wnt-4 and Wnt-11, and by misexpressing it with a retrovirus vector in the limb bud. Wnt-5a expression is detected in the limb-forming region at stage 14, and in the apical ectodermal ridge and distal mesenchyme of the limb bud. The signal was graded along the proximal-distal axis at stages 20-28 and also along the anterior-posterior axis during early stages. It disappeared in the cartilage-forming region after stage 26, and was restricted to the region surrounding the phalanges at stage 34. Wnt-4 and Wnt-11, other members of the Wnt-5a-subclass, were expressed with a distinct spatiotemporal pattern during the later phase. Wnt-4 was expressed in the articular structure and Wnt-11 was expressed in the dorsal and ventral mesenchyme adjacent to the ectoderm. Wnt-5a expression was partially reduced after apical ectodermal ridge removal, whereas Wnt-11 expression was down-regulated by dorsal ectoderm removal. Therefore, expression of these Wnt was differentially regulated by the ectodermal signal. Misexpression of Wnt-5a in the limb bud with the retrovirus resulted in truncation of long bones predominantly in the zeugopod because of retarded chondrogenic differentiation. Distal elements, such as the phalanges and metacarpals, were not significantly reduced in size. These results suggest that Wnt-5a is involved in pattern formation along the proximal-distal axis by regulation of chondrogenic differentiation.     

Scanning electron microscopy of Echinostoma revolutum (Trematoda) during development in the chick embryo and the domestic chick

Fried B. and Fujino T. 1984. Scanning electron microscopy of Echinostoma revolutum (Trematoda) during development in the chick embryo and the domestic chick. International Journal for Parasitology14: 75–81. Scanning electron microscopy (SEM) was used to study the development of chemically excysted metacercariae of Echinostoma revolutum on the chick chorioallantois. SEM studies were also made on preovigerous adults of E. revolutum grown in the domestic chick. During worm development on the chorioallantois the tegument changed from smooth to granular and sensory papillae on the suckers became well-defined. As worms developed on the chorioallantois the cephalic collar spines became thicker and more curved and the tegumentary spines showed marked changes in shape, size and distribution on both ventral and dorsal aspects of the body. Changes in the surface ultrastructure of worms grown on the chorioallantois were essentially similar to those observed in preovigerous worms from chicks.     

Beta-catenin-dependent Wnt signaling in apical ectodermal ridge induction and FGF8 expression in normal and limbless mutant chick limbs

The fibroblast growth factor (FGF) and beta-catenin-dependent Wnt signaling pathways are key regulators of vertebrate limb development. FGF10 induces expression of Wnt3a, which regulates the formation and FGF8 expression of the apical ectodermal ridge (AER). In amelic limbless limbs, an AER fails to form and FGF8 is not expressed, despite expression of FGF10. It has been found that Wnt3a is initially expressed in limbless ectoderm, although subsequently is drastically reduced. In addition, changes in the expression pattern or level of several Frizzled receptors, Axin, Lef1/Tcf1 and beta-catenin have been found in limbless limbs. Notably, while normal wing buds respond to LiCl-stimulated activation of beta-catenin-dependent signaling by forming ectopic, FGF8-expressing AER, LiCl was unable to induce an AER in limbless wing buds. The results of this study suggest that the limbless gene is required for beta-catenin-dependent Wnt signaling in limb ectoderm leading to FGF8 expression and AER formation.     

Fibroblast growth factor-induced gene expression and cartilage pattern formation in chick limb bud recombinants

To clarify the roles of fibroblast growth factors (FGF) in limb cartilage pattern formation, the effects of various FGF on recombinant limbs that were composed of dissociated and reaggregated mesoderm and ectodermal jackets were examined. Fibroblast growth factor-soaked beads were inserted just under the apical ectodermal ridge (AER) of recombinant limbs and the recombinant limbs were grafted and allowed to develop. Control recombinant limbs without FGF beads formed one or two cartilage elements. Recombinants with FGF-4 beads formed up to five cartilage elements, which were aligned along the anteroposterior (AP) axis. Each cartilage element showed digit-like segmentation. In contrast, recombinants with FGF-2 beads showed formation of multiple thick and unsegmented cartilage rods, which elongated inside and outside the AP plane from the distal end of the recombinants. Recombinants with FGF-8 beads formed a truncated cartilage pattern and recombinants with FGF-10 beads formed a cartilage pattern similar to that of the control recombinants. The expression of the Fgf-8, Msx-1 and Hoxa-13 genes in the developing recombinant limbs were examined. FGF-4 induced extension of the length of the Fgf-8-positive epidermis, or AER, along the AP axis 5 days after grafting, at which time the digits are specified. FGF-2 induced expansion of the Msx-1-positive area, first in the proximal direction and then along the dorsoventral axis. The functions of these FGF in recombinant and normal limb patterning are discussed in this paper.     

Isolation and characterization of mRNAs accumulated during in-vitro flower bud formation

The development of vegetative and generative buds on thin-layer expiants of tobacco (Nicotiana tabacum L. cv. Samsun) has been studied at the level of translatable mRNA to detect changes in the mRNA population during bud initiation and differentiation, and several quantitative differences were found. By differential screening of a cDNA library obtained from flower-bud-regenerating explants we have isolated a group of six cDNA clones representing genes that are preferentially expressed during in-vitro flower bud formation. Nucleotide sequence analysis of one of these cDNAs, pAP8, showed that the most likely open reading frame has some typical characteristics of, and homology with, extensin-like genes. Northern blot analysis and in-situ hybridization suggest a specific role for these extensin-like genes in flower bud initiation on tobacco pedicel explants.     

Microfibril formation in chick notochordal cells

The central parts of the chick notochord at Hamburger and Hamilton's stages 20–22 were investigated by electron microscopy. Electron-dense bodies of various sizes and shapes and bounded by a limiting membrane were found in the central cells of the notochord. These dense bodies contained fibrous material or microfibrils which ranged from 120 to 600 Å in diameter. The large microfibrils often exhibited a typical repeating period with an interval of about 320 Å. These dense bodies were always located near the cell membrane, which is rough or irregular in the central parts of the notochord at these stages. The fibrous core material of the dense body frequently shows striking similarities to amorphous fibrous material in the intercellular space of the central parts of the notochord, where they are situated at a considerable distance from the perinotochordal sheath space. From these results, it seems reasonable to suggest that the central cells as well as the peripheral cells of the notochord are capable of forming microfibrils similar to those observed in the perinotochordal sheath space.Moreover, they may play an important role in the total fibrillogenesis of the notochord.     

Gene manipulation of chick embryos in vitro, early chick culture, and long survival in transplanted eggs

We introduce a revolutionary gene transfer system in chick: transfect chick embryos at early developmental stage by electroporation in vitro, Early Chick (EC) culture, and transplant to the egg to let the embryo survive until E5.5. Referring to the fate map, we could target the tissues of transfection, or transfect large areas of the embryo. We could get tissue-specific expression of a transgene by tissue-specific promoter. This method is very convenient and rapid, but allows us to get stable expression of the transgene in combination with transposon system.