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.

Identification and distribution of gap junctions in the mesoderm of the developing chick limb bud

Development ◽  
1978 ◽  
Vol 46 (1) ◽  
pp. 99-110
Author(s):  
Robert O. Kelley ◽  
John F. Fallon
Keyword(s):  
Electron Microscopy ◽  
Gap Junctions ◽  
Limb Development ◽  
Freeze Fracture ◽  
Mesenchymal Cells ◽  
Limb Bud ◽  
Metabolic Coupling ◽  
Transmission Electron ◽  
Communicating Junctions ◽  
Cell Processes

Sub-ridge, core, anterior and posterior borders of mesoderm were dissected from stages 22–24 chick wing buds to investigate whether structures for intercellular coupling develop between mesenchymal cells. Fine structure was examined using techniques of transmission electron microscopy, freeze-fracture and scanning electron microscopy. Gap (communicating) junctions which were observed between mesenchymal cells of all limb bud regions were distributed between apposed cell bodies, points of contact between cell processes and other cell bodies, and between contacting tips of slender cell projections. In addition, particularly in the subridge region, filopodia were observed to extend through the intercellular matrix to contact other cells several micrometers distant. The observations reported in this paper show that mesodermal cells throughout the limb have the structural capability for electrotonic and metabolic coupling during a critical period of morphogensisis in the avian limb. Whether intercellular signals which are thought to be transmitted through gap junctions are active in normal limb development remains to be investigated.

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 chick limbless mutation causes abnormalities in limb bud dorsal-ventral patterning: implications for the mechanism of apical ridge formation

Development ◽  
1996 ◽  
Vol 122 (12) ◽  
pp. 3851-3861 ◽  
Author(s):  
U. Grieshammer ◽  
G. Minowada ◽  
J.M. Pisenti ◽  
U.K. Abbott ◽  
G.R. Martin
Keyword(s):  
Gene Expression ◽  
Limb Development ◽  
Positional Information ◽  
Limb Bud ◽  
Local Application ◽  
Chick Embryos ◽  
Limb Buds ◽  
Early Stages ◽  
Potential Link

In chick embryos homozygous for the limbless mutation, limb bud outgrowth is initiated, but a morphologically distinct apical ridge does not develop and limbs do not form. Here we report the results of an analysis of gene expression in limbless mutant limb buds. Fgf4, Fgf8, Bmp2 and Msx2, genes that are expressed in the apical ridge of normal limb buds, are not expressed in the mutant limb bud ectoderm, providing molecular support for the hypothesis that limb development fails in the limbless embryo because of the inability of the ectoderm to form a functional ridge. Moreover, Fgf8 expression is not detected in the ectoderm of the prospective limb territory or the early limb bud of limbless embryos. Since the early stages of limb bud outgrowth occur normally in the mutant embryos, this indicates that FGF8 is not required to promote initial limb bud outgrowth. In the absence of FGF8, Shh is also not expressed in the mutant limb buds, although its expression can be induced by application of FGF8-soaked beads. These observations support the hypothesis that Fgf8 is required for the induction of Shh expression during normal limb development. Bmp2 expression was also not detected in mutant limb mesoderm, consistent with the hypothesis that SHH induces its expression. In contrast, SHH is not required for the induction of Hoxd11 or Hoxd13 expression, since expression of both these genes was detected in the mutant limb buds. Thus, some aspects of mesoderm A-P patterning can occur in the absence of SHH and factors normally expressed in the apical ridge. Intriguingly, mutant limbs rescued by local application of FGF displayed a dorsalized feather pattern. Furthermore, the expression of Wnt7a, Lmx1 and En1, genes involved in limb D-V patterning, was found to be abnormal in mutant limb buds. These data suggest that D-V patterning and apical ridge formation are linked, since they show that the limbless mutation affects both processes. We present a model that explains the potential link between D-V positional information and apical ridge formation, and discuss the possible function of the limbless gene in terms of this model.

Ultrastructural identification of extracellular matrix and cell surface components during limb morphogenesis in man

Development ◽  
1975 ◽  
Vol 34 (1) ◽  
pp. 1-18
Author(s):  
Robert O. Kelley
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Basal Lamina ◽  
Mesenchymal Cell ◽  
Ruthenium Red ◽  
Human Hand ◽  
Binding Characteristics ◽  
Limb Morphogenesis ◽  
Testicular Hyaluronidase

Development of the human hand plate (stages 16–17) has been analyzed with emphasis on differentiation of elements within the extracellular matrix and the composition of the mesenchymal cell surface. The epithelial—mesenchymal interface contains a basal lamina and a sublaminar matrix exhibiting: (a) collagen fibrils with characteristic 63–64 nm banding; (b) non-banded filaments, 10–15 nm in diameter; (c) ruthenium red-positive particles, 12–15 nm in diameter; and (d) attenuated threads, 3·5–5·0 nm in diameter which interconnect particles, fibrils, filaments and the basal lamina. Processes of mesenchymal cells penetrate this matrix network. In addition to staining with ruthenium red, components of basal laminae bind to ferritin-conjugated Concanavalin A, greatest binding being localized on the mesenchymal surface of the lamina. Asymmetry of binding is removed by incubation of exposed laminae with trypsin (5 µg/ml). Regional differences in these staining and binding characteristics within the subepithelial matrix have not been observed in the hand plate. However, precartilaginous extracellular zones deep within the plate are notably unstructured in comparison to the sublaminar region. Ruthenium red-positive materials at mesenchymal cell surfaces display sensitivity to testicular hyaluronidase, Pronase and trypsin but resist removal with neuraminidase and EDTA. These features of the substrate in situ may be important in the regulation of mesenchymal cell behavior during limb morphogenesis in man.

The role of morphogenetic cell death during abnormal limb-bud outgrowth in mice heterozygous for the dominant mutation Hemimelia-extra toe (Hmx)

Development ◽  
1981 ◽  
Vol 65 (Supplement) ◽  
pp. 289-307
Author(s):  
T. B. Knudsen ◽  
D. M. Kochhar
Keyword(s):  
Basal Lamina ◽  
Developmental Stages ◽  
Mesenchymal Cell ◽  
Limb Bud ◽  
Dominant Mutation ◽  
Limb Malformations ◽  
Congenital Limb ◽  
Filamentous Material ◽  
Limb Pattern

A dominant mutation in the mouse, Hemimelia-extra toe (Hmx), induces congenital limb malformations in heterozygotes. Typical expression includes axial shortening of the radius, tibia and talus (‘hemimelia’), with supernumerary metacarpals, metatarsals, and digits (‘polydactyly’). Pathogenesis was investigated during developmental stages 16 through 22 (1lth through 15th days of gestation). Full expression was apparent during stage 20 when the limb pattern was comprised of pre-cartilaginous anlagen. Formation of a pre-axial protrusion on the autopod during stage 17 or 18 was the earliest gross abnormality, and foreshadowed the development of supernumerary digits. Microscopically, there was an alteration in the pattern of physiologic cellular degeneration (PCD) programmed to occur within the zeugopod and autopod. The ‘opaque patch’ (mesodermal necrotic zone normally occurring between tibial and fibular anlagen) was overextended pre-axially causing resorption of the tibial precartilage. Additionally, PCD normally occurring within the basal cell layer of the apical ectodermal ridge (AER) and the ‘foyer primaire préaxial’ was not expressed in the mutant autopod. This occurred in association with outgrowth of the protrusion. The pre-axial portion of the AER remained in an abnormally thickened, viable, proliferative state, and did not undergo scheduled degression. This may have been the basis for prolonged induction of pre-axial outgrowth. Paucity of mesenchymal cell filopodial processes extended along the basal lamina, as well as a rarefaction of the filamentous material normally associated with the mesodermal face of the basal lamina, was detected at the pre-axial AER-mesenchymal interface on stage 18. A potential involvement of epithelial-mesenchymal interactions in the induction of epithelial PCD is discussed.

Apical ridge dependent and independent mesodermal domains of GHox-7 and GHox-8 expression in chick limb buds

Development ◽  
1992 ◽  
Vol 116 (3) ◽  
pp. 811-818 ◽  
Author(s):  
M.A. Ros ◽  
G. Lyons ◽  
R.A. Kosher ◽  
W.B. Upholt ◽  
C.N. Coelho ◽  
...  
Keyword(s):  
Pattern Formation ◽  
Limb Development ◽  
Apical Ectodermal Ridge ◽  
Limb Bud ◽  
Rapid Decline ◽  
Limb Buds ◽  
Limb Outgrowth ◽  
Removal Experiments ◽  
The Relationship

The homeobox-containing genes GHox-7 and GHox-8 have been proposed to play fundamental roles in limb development. The expression of GHox-8, by the apical ridge cells, and GHox-7, in the subridge mesoderm, suggests the involvement of these two genes in limb outgrowth and proximo-distal pattern formation. A straightforward way to test this is to remove the apical ridge. Here we report the relationship between the mesodermal expression of GHox-7 and GHox-8 and the apical ectodermal ridge in the chick limb bud. The data from ridge removal experiments indicate that there are at least two domains of GHox-7 expression in the apical limb bud mesoderm. The posterior subridge GHox-7 domain in the progress zone requires the influence of the apical ridge for continued expression, while the anterior GHox-7 domain continues expression after ridge removal. Posterior subridge mesoderm is exquisitely sensitive to the loss of the ridge in that GHox-7 expression by these cells is reduced in only two hours and undetectable by three hours after ridge removal. It would appear that one of the ways progress zone cells respond to the apical ridge signal is by expressing GHox-7. The loss of ridge influence whether by growth at the apex or by ridge removal is followed by an unusually rapid decline in detectable GHox-7 transcripts. Maintenance of GHox-8 expression by the anterior mesoderm appears to be independent of the presence of the apical ridge.(ABSTRACT TRUNCATED AT 250 WORDS)

Observations of Dependent Histogenesis in Salamander Limb Development

Development ◽  
1963 ◽  
Vol 11 (2) ◽  
pp. 325-338
Author(s):  
Cyril V. Finnegan
Keyword(s):  
Limb Development ◽  
Cell Mass ◽  
Limb Bud ◽  
Previous Experience ◽  
Limb Buds ◽  
Self Differentiation ◽  
Positive Results

Previous work in this laboratory (Finnegan, 1962) had suggested that limb development might be specifically enhanced by experimentally associated somite mesoderm tissue. Detwiler (1938), Swett (1945) and Nicholas (1958) called attention to the influence of this mesoderm in the production of duplications when limb buds were transplanted heterotopically to the superficial somite region and, more recently, Amano (1960) stated that somite tissue was required, inductively and materially, for limb development. In an analysis of development it is assumed that a group of cells, whose histogenesis has been determined by their previous experience, will evidence that histogenesis if placed in an environment in which they continue to develop. Thus, after limb bud transplantation to the flank in urodeles, positive results (that is, histogenesis, proximally, accompanied by growth, distally) have been interpreted as self-differentiation of the limb bud cell mass (see review by Nicholas, 1955).

Expression of chicken fibroblast growth factor homologous factor (FHF)-1 and of differentially spliced isoforms of FHF-2 during development and involvement of FHF-2 in chicken limb development

Development ◽  
1999 ◽  
Vol 126 (2) ◽  
pp. 409-421
Author(s):  
I. Munoz-Sanjuan ◽  
B.K. Simandl ◽  
J.F. Fallon ◽  
J. Nathans
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Neural Tube ◽  
Limb Development ◽  
Limb Bud ◽  
Fibroblast Growth ◽  
Limb Buds ◽  
Adjacent Structures ◽  
Morphological Defects ◽  
Zone Of Polarizing Activity

Members of the fibroblast growth factor (FGF) family have been identified as signaling molecules in a variety of developmental processes, including important roles in limb bud initiation, growth and patterning. This paper reports the cloning and characterization of the chicken orthologues of fibroblast growth factor homologous factors-1 and −2 (cFHF-1/cFGF-12 and cFHF-2/cFGF-13, respectively). We also describe the identification of a novel, conserved isoform of FHF-2 in chickens and mammals. This isoform arises by alternative splicing of the first exon of the FHF-2 gene and is predicted to encode a polypeptide with a distinct amino-terminus. Whole-mount in situ hybridization reveals restricted domains of expression of cFHF-1 and cFHF-2 in the developing neural tube, peripheral sensory ganglia and limb buds, and shows that the two cFHF-2 transcript isoforms are present in non-overlapping spatial distributions in the neural tube and adjacent structures. In the developing limbs, cFHF-1 is confined to the posterior mesoderm in an area that encompasses the zone of polarizing activity and cFHF-2 is confined to the distal anterior mesoderm in a region that largely overlaps the progress zone. Ectopic cFHF-2 expression is induced adjacent to grafts of cells expressing Sonic Hedgehog and the zone of cFHF-2 expression is expanded in talpid2 embryos. In the absence of the apical ectodermal ridge or in wingless or limbless mutant embryos, expression of cFHF-1 and cFHF-2 is lost from the limb bud. A role for cFHF-2 in the patterning and growth of skeletal elements is implied by the observation that engraftment of developing limb buds with QT6 cells expressing a cFHF-2 isoform that is normally expressed in the limb leads to a variety of morphological defects. Finally, we show that a secreted version of cFHF-2 activates the expression of HoxD13, HoxD11, Fgf-4 and BMP-2 ectopically, consistent with cFHF-2 playing a role in anterior-posterior patterning of the limb.

Vascular regression during the formation of the free digits in the avian limb bud: a comparative study in chick and duck embryos

Development ◽  
1985 ◽  
Vol 85 (1) ◽  
pp. 239-250
Author(s):  
J. M. Hurle ◽  
E. Colvee ◽  
M. A. Fernandez-Teran
Keyword(s):  
Cell Death ◽  
Blood Vessels ◽  
Mesenchymal Cell ◽  
Limb Bud ◽  
Death Process ◽  
Electron Microscopic ◽  
Transmission Electron ◽  
Transmission Electron Microscopic ◽  
Microscopic Studies ◽  
Cell Death Process

The pattern and structure of the blood vessels of the interdigital spaces of the leg bud have been studied by means of Indian ink injections and transmission electron microscopy in the chick and duck embryos. The results show that in the chick the interdigital necrotic process responsible for the freeing of the digits is followed by regression of the blood vessels. In the webbed foot of the duck, the interdigital necrotic processes are not followed by vascular regression. Transmission electron microscopic studies show that both in the chick and in the duck, interdigital blood vessels are immature structures lacking basal lamina. Dead cells of presumably endothelial origin were detected in the lumen of the regressing blood vessels of the chick but not in the duck. However, the intensity of this cell death process does not appear to be high enough to account by itself for the disappearance of the interdigital blood vessels. The possible relationships between interdigital mesenchymal cell death and vascular regression are discussed.

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