Electron Microscopic Observations on the Basal Lamina of Chick Limb Buds After Trypsin and Edta Treatment

1968 ◽  
Vol 3 (3) ◽  
pp. 373-380
Author(s):  
S. C. GOEL ◽  
A. JURAND
Keyword(s):  
Basal Lamina ◽  
Salt Solution ◽  
Limb Development ◽  
Hind Limb ◽  
Physiological Role ◽  
Electron Microscopic ◽  
Limb Buds ◽  
Edta Treatment ◽  
Subsequent Separation ◽  
Calcium And Magnesium

Chick hind-limb buds were treated first with calcium- and magnesium-free balanced salt solution and then with either trypsin or EDTA. Treatment with these chemicals, and the subsequent separation of the ectodermal covering from the mesoderm, produces two different results as regards the basal lamina. In the case of trypsin, the lamina stays with the ectoderm but shows signs of probable tryptic digestion. After EDTA treatment the lamina stays with the mesoderm and becomes very loose and swollen. This loosening allows the structure of the otherwise compact lamina to be resolved so that banded collagen fibrils are seen participating in its substance. The above observations are discussed in relation to the mode of action of the two chemicals. The physiological role and the origin of the basal lamina, as well as the possible implications of the present findings to the question of limb development and morphogenesis, are also discussed. A note has also been made regarding the various terms used to describe the epitheliomesenchymal junction layers.

Fine structural analysis of limb development in the wingless mutant chick embryo

Development ◽  
1982 ◽  
Vol 68 (1) ◽  
pp. 69-86
Author(s):  
Linwood M. Sawyer
Keyword(s):  
Gap Junctions ◽  
Basal Lamina ◽  
Limb Development ◽  
Mesenchymal Cell ◽  
Ruthenium Red ◽  
Limb Bud ◽  
Normal Embryo ◽  
Limb Buds ◽  
Transmission Electron ◽  
Dense Vesicles

The fine structure of the normal and wingless chick limb bud was examined with scanning and transmission electron microscopy. The apical ectodermal ridge (AER) of the normal limb bud was composed of pseudostratined columnar cells. These cells contained gap junctions, electron-dense vesicles, and numerous microtubules and microfilaments that were oriented perpendicularly to the basal lamina. Microfilaments were also found coursing transversely in the basal cell cytoplasm. The ectoderm of the wingless mutant limb bud lacked a well-developed AER and resembled the dorsal and ventral ectoderm of the normal embryo. Gap junctions and electron-dense vesicles found in the AER of the normal limb bud were not apparent in the mutant ectoderm. The normal-limb bud mesoderm is composed of stellate cells that are oriented at right angles to the overlying ectoderm. There is a prominent subectodermal space that is traversed by numerous mesenchymal cell filopodia. The mesodermal cells of the mutant limb bud are compact and round and have short stubby filopodia, while the cells of the adjacent flank mesoderm are stellate. The subectodermal space is absent and the mesodermal cells are in intimate association with the basal lamina of the overlying ectoderm. Ruthenium red was employed as an extracellular marker for glycosaminoglycan$. No differences were found in the distribution of these substances in normal and mutant limb buds. In severalcases the basal lamina of the mutant limb bud ectoderm was discontinuous aqd the lamina lucida wasnot apparent. The results indicate that the mutation has an effect on the limb buds' ability to maintain a well-developed AER and basal lamina. It also suggest$ that the wingless gene affects the shape and possibly the mobility of the limb-bud mesoderm cells.

Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds

Development ◽  
1993 ◽  
Vol 119 (1) ◽  
pp. 247-261 ◽  
Author(s):  
B.A. Parr ◽  
M.J. Shea ◽  
G. Vassileva ◽  
A.P. McMahon
Keyword(s):  
Limb Development ◽  
Expression Patterns ◽  
Limb Buds ◽  
Expression Studies ◽  
The Family ◽  
The Central Nervous System ◽  
Discrete Domains ◽  
Early Developmental ◽  
The Brain

Mutation and expression studies have implicated the Wnt gene family in early developmental decision making in vertebrates and flies. In a detailed comparative analysis, we have used in situ hybridization of 8.0- to 9.5-day mouse embryos to characterize expression of all ten published Wnt genes in the central nervous system (CNS) and limb buds. Seven of the family members show restricted expression patterns in the brain. At least three genes (Wnt-3, Wnt-3a, and Wnt-7b) exhibit sharp boundaries of expression in the forebrain that may predict subdivisions of the region later in development. In the spinal cord, Wnt-1, Wnt-3, and Wnt-3a are expressed dorsally, Wnt-5a, Wnt-7a, and Wnt-7b more ventrally, and Wnt-4 both dorsally and in the floor plate. In the forelimb primordia, Wnt-3, Wnt-4, Wnt-6 and Wnt-7b are expressed fairly uniformly throughout the limb ectoderm. Wnt-5a RNA is distributed in a proximal to distal gradient through the limb mesenchyme and ectoderm. Along the limb's dorsal-ventral axis, Wnt-5a is expressed in the ventral ectoderm and Wnt-7a in the dorsal ectoderm. We discuss the significance of these patterns of restricted and partially overlapping domains of expression with respect to the putative function of Wnt signalling in early CNS and limb development.

Tetrazolium Salt and Electron-Microscopic Studies of Cellular Degeneration and Necrosis in the Interdigital Areas of the Developing Chick Limb

1971 ◽  
Vol 8 (1) ◽  
pp. 229-251
Author(s):  
S. P. HAMMAR ◽  
N. K. MOTTET
Keyword(s):  
Cell Death ◽  
Enzyme Activity ◽  
Succinate Dehydrogenase ◽  
Hind Limb ◽  
Morphological Changes ◽  
Morphological Evidence ◽  
Tetrazolium Salt ◽  
Limb Buds ◽  
Cellular Degeneration ◽  
Intracellular Digestion

Cellular degeneration and necrosis were studied in the interdigital areas of developing hind limb buds of normal chick embryos by means of enzyme-specific tetrazolium salts and electron microscopy. Using succinic acid as a substrate and the tetrazolium salt, nitro blue tetrazolium, succinate dehydrogenase was specifically demonstrated via a colour reaction in which degenerating and necrotic cells--those with no enzyme activity--did not stain, while those with enzyme activity stained deeply blue-black. The interdigital cells exhibited near absent levels of succinate dehydrogenase as early as stage 26-27, one to two days prior to when morphological evidence of degeneration and necrosis was present. It was postulated that the mechanism of cell death resulted from decreased activity and/or loss of strategic cellular enzymes such as succinate dehydrogenase, with a subsequent fall in the cellular adenosine triphosphate (ATP) level and a resultant compromise in vital cellular processes, eventually leading to cell death. The evidence indicated that ‘biochemical degeneration’ occurred prior to morphological changes in cells. The ultrastructural events of degenerating and necrotic cells were also studied. Most ‘dying’ cells observed had already been phagocytosed and were observed in various stages of degeneration. The degenerating cells exhibited both nuclear and cytoplasmic changes. There was evidence of active intracellular digestion within the phagocytes. Numerous lysosomes were observed within these cells, and some appeared fused with the digestive vacuole's membranes. The origin of the phagocytes was not determined. No definite information was obtained concerning the utility of cellular degeneration and necrosis in the interdigital areas of the hind limb buds. It appeared to be at least partially causal in separation of the digits.

Branching morphogenesis in the avian lung: electron microscopic studies using cationic dyes

Development ◽  
1986 ◽  
Vol 94 (1) ◽  
pp. 189-205
Author(s):  
Betty C. Gallagher
Keyword(s):  
Tannic Acid ◽  
Basal Lamina ◽  
Branching Morphogenesis ◽  
Ruthenium Red ◽  
Interparticle Spacing ◽  
Blue Staining ◽  
Mouse Lung ◽  
Alcian Blue Staining ◽  
Electron Microscopic ◽  
Avian Lung

The developing chick lung was examined in the electron microscope for intimate cell contacts between epithelium and mesenchyme, discontinuities in the basal lamina and substructure of the basement membrane. Cell filopodia were seen which crossed the basal lamina from both the epithelial and the mesenchymal cells. Ruthenium red and tannic acid staining of the basal lamina of the chick lung showed it to be thin and sometimes discontinuous at the tips compared to the more substantial basal lamina in the interbud areas. The bilaminar distribution of particles seen with ruthenium red is similar to those seen in the cornea and lens. With tannic acid staining, filaments could be seen which crossed the lamina lucida and connected with the lamina densa. Spikes perpendicular to the basal lamina were sometimes seen with a periodicity of approximately 110 nm. Alcian blue staining revealed structure similar to that seen by ruthenium red staining in the salivary and mammary glands, although the interparticle spacing was closer. Collagen was located in areas of morphogenetic stability, as has been seen by other investigators in different tissues. Collagen was coated with granules (probably proteoglycan) at periodic intervals when stained with ruthenium red. The fibrils were oriented circumferentially around the mesobronchus and were assumed to continue into the bud, but the fibres curve laterally at the middle of a bud. This orientation is opposite to that seen by another investigator in the mouse lung. In general, the observations made in the avian lung are similar to those seen in branching mammalian tissue. It is likely, therefore, that the chick lung uses strategies in its morphogenesis that are similar to those that have been elucidated previously in developing mammalian organs.

Role of the chicken homeobox-containing genes GHox-4.6 and GHox-8 in the specification of positional identities during the development of normal and polydactylous chick limb buds

Development ◽  
1992 ◽  
Vol 115 (2) ◽  
pp. 629-637 ◽  
Author(s):  
C.N. Coelho ◽  
W.B. Upholt ◽  
R.A. Kosher
Keyword(s):  
Limb Development ◽  
Ectopic Expression ◽  
Limb Bud ◽  
Chick Embryos ◽  
Limb Buds ◽  
Hox Gene ◽  
Wing Bud ◽  
Coated Bead ◽  
Experimentally Induced

During early stages of normal chick limb development, the homeobox-containing (HOX) gene GHox-4.6 is expressed throughout the posterior mesoderm of the wing bud from which most of the skeletal elements including the digits will develop, whereas GHox-8 is expressed in the anterior limb bud mesoderm which will not give rise to skeletal elements. In the present study, we have examined the expression of GHox-4.6 and GHox-8 in the wing buds of two polydactylous mutant chick embryos, diplopodia-5 and talpid2, from which supernumerary digits develop from anterior limb mesoderm, and have also examined the expression of these genes in response to polarizing zone grafts and retinoic acid-coated bead implants which induce the formation of supernumerary digits from anterior limb mesoderm. We have found that the formation of supernumerary digits from the anterior mesoderm in mutant and experimentally induced polydactylous limb buds is preceded by the ectopic expression of GHox-4.6 in the anterior mesoderm and the coincident suppression of GHox-8 expression in the anterior mesoderm. These observations suggest that the anterior mesoderm of the polydactylous limb buds is “posteriorized” and support the suggestion that GHox-8 and GHox-4.6, respectively, are involved in specifying the anterior non-skeletal and posterior digit-forming regions of the limb bud. Although the anterior mesodermal domain of GHox-8 expression is severely impaired in the mutant and experimentally induced polydactylous limb buds, this gene is expressed by the prolonged, thickened apical ectodermal ridges of the polydactylous limb buds that extend along the distal anterior as well as the distal posterior mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)

Sonic hedgehog is not required for polarising activity in the Doublefoot mutant mouse limb bud

Development ◽  
1998 ◽  
Vol 125 (3) ◽  
pp. 351-357 ◽  
Author(s):  
C. Hayes ◽  
J.M. Brown ◽  
M.F. Lyon ◽  
G.M. Morriss-Kay
Keyword(s):  
Gene Expression ◽  
Limb Development ◽  
Target Genes ◽  
Anterior Part ◽  
Expression Patterns ◽  
Ectopic Expression ◽  
Mutant Gene ◽  
Limb Bud ◽  
Active Constituent ◽  
Limb Buds

The mouse mutant Doublefoot (Dbf) shows preaxial polydactyly of all four limbs. We have analysed limb development in this mutant with respect to morphogenesis, gene expression patterns and ectopic polarising activity. The results reveal a gain-of-function mutation at a locus that mediates pattern formation in the developing limb. Shh expression is identical with that of wild-type embryos, i.e. there is no ectopic expression. However, mesenchyme from the anterior aspects of Dbf/+ mutant limb buds, when transplanted to the anterior side of chick wing buds, induces duplication of the distal skeletal elements. Mid-distal mesenchymal transplants from early, but not later, Dbf/+ limb buds are also able to induce duplication. This demonstration of polarising activity in the absence of Shh expression identifies the gene at the Dbf locus as a new genetic component of the Shh signalling pathway, which (at least in its mutated form) is able to activate signal transduction independently of Shh. The mutant gene product is sufficient to fulfil the signalling properties of Shh including upregulation of the direct Shh target genes Ptc and Gli, and induction of the downstream target genes Bmp2, Fgf4 and Hoxd13. The expression domains of all these genes extend from their normal posterior domains into the anterior part of the limb bud without being focused on a discrete ectopic site. These observations dissociate polarising activity from Shh gene expression in the Dbf/+ limb bud. We suggest that the product of the normal Dbf gene is a key active constituent of the polarising region, possibly acting in the extracellular compartment.

The genesis of membrane bone in the embryonic chick maxilla: epithelial-mesenchymal tissue recombination studies

Development ◽  
1980 ◽  
Vol 56 (1) ◽  
pp. 269-281
Author(s):  
Mary S. Tyler ◽  
David P. McCobb
Keyword(s):  
Bone Formation ◽  
Hind Limb ◽  
Bone Grafts ◽  
Chick Embryos ◽  
Embryonic Chick ◽  
Limb Buds ◽  
Mesenchymal Tissue ◽  
Membrane Bone ◽  
Tissue Recombination

In the present study, the question of whether a relatively non-specific epithelial requirement exists for membrane bone formation within the maxillary mesenchyme was investigated. Organ rudiments from embryonic chicks of three to five days of incubation (HH 18–25) were enzymatically separated into the epithelial and mesenchymal components. Maxillarymesenchyme (from embryos HH 18–19) which in the absence of epithelium will not form bone was recombined with epithelium from maxillae of similarly aged embryos (homotypichomochronic recombination) and of older embryos (HH 25) (homotypic-heterochronicrecombination). Heterotypic recombinations were made between maxillary mesenchyme (HH 18–19) and the epithelium from wing and hind-limb buds (HH 19–22). Recombinants were grown as grafts on thechorioallantoic membranes of host chick embryos. Grafts of intact maxillae, isolated maxillary mesenchyme, and isolated epithelia from the maxilla, wing-, and hind-limb buds weregrown as controls. The histodifferentiation of grafted intact maxillae was similar to that in vivo; both cartilage and membrane bone differentiated within the mesenchyme. Grafts of maxillary mesenchyme (from embryos HH 18–19) grown in the absence of epithelium formed cartilage but did not form membrane bone. Grafts of maxillary mesenchyme (from embryos HH 18–19) recombined with epithelium in homotypichomochronic, homotypic-heterochronic, and heterotypic tissue combinations formed membrane bone in addition to cartilage. These results indicate that maxillary mesenchyme requires the presence of epithelium to promote osteogenesis and that this epithelial requirement is relatively non-specific in terms of type and age of epithelium.

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