Strategies to detect interdigital cell death in the frog, Xenopus laevis: T3 accerelation, BMP application, and mesenchymal cell cultivation

In the present work, we have analysed the possible involvement of ectodermal tissue in the control of interdigital mesenchymal cell death. Two types of experiments were performed in the stages previous to the onset of interdigital cell death: (i) removal of the AER of the interdigit; (ii) removal of the dorsal ectoderm of the interdigit. After the operation embryos were sacrificed at 10–12h intervals and the leg buds were studied by whole-mount cartilage staining, vital staining with neutral red and scanning electron microscopy. Between stages 27 and 30, ridge removal caused a local inhibition of the growth of the interdigit. In a high percentage of the cases, ridge removal at these stages was followed 30–40 h later by the formation of ectopic nodules of cartilage in the interdigit. The incidence of ectopic cartilage formation was maximum at stage 29 (60%). In all cases, cell death took place on schedule although the intensity and extent of necrosis appeared diminished in relation to the intensity of inhibition of interdigital growth and to the presence of interdigital cartilages. Ridge removal at stage 31 did not cause inhibition of the growth of the interdigit and ectopic chondrogenesis was only detected in 3 out of 35 operated embryos. Dorsal ectoderm removal from the proximal zone of the interdigit at stage 29 caused the chondrogenesis of the proximal interdigital mesenchyme in 6 out of 18 operated embryos. The pattern of neutral red vital staining was consistent with these results revealing a partial inhibition of interdigital cell death in the proximal zone of the interdigit. It is proposed that under the present experimental conditions the mesenchymal cells are diverted from the death programme by a primary transformation into cartilage.

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Genetic inhibition of mesenchymal cell death and the development of form and skeletal pattern in the limbs of talpid3 (ta3) mutant chick embryos

Development ◽

10.1242/dev.31.3.747 ◽

1974 ◽

Vol 31 (3) ◽

pp. 747-760

Author(s):

J. R. Hinchliffe ◽

P. V. Thorogood

Keyword(s):

Cell Death ◽

Soft Tissue ◽

Limb Development ◽

Chondroitin Sulphate ◽

Vital Staining ◽

Mesenchymal Cell ◽

Equivalent Position ◽

Skeletal Pattern ◽

Mesenchyme Cells ◽

Interdigital Cell Death

Vital staining reveals that in homozygous (ta3/ta3) talpid3 embryos, the areas of mesenchymal cell death which occur regularly in normal limb development are absent or reduced. The necrotic locus in the central mesenchyme (the ‘opaque patch’) which in the normal chick limb reaches maximum development at stages 24 and 25 (4½–5 days) is absent or much reduced in talpid3 fore- and hindlimb-buds. Autoradiographic studies, following application of a 2 h pulse of 40 μCi of 35SO4 to the vitelline circulation, show that normal tibia and fibula incorporate 35SO4 into chondroitin sulphate at stage 24 and more strongly at stage 26 during the process of chondrogenesis. The mesenchyme in the opaque patch region of normal limbs ceases to incorporate 35SO4 into chondroitin sulphate at stage 24. Talpid3 mesenchyme cells in the equivalent position at stages 24 and 26 continue to incorporate 35SO4, remain viable and become chondrogenic. It is suggested that absence or reduction of this central necrotic locus in talpid3 is causally related to the fusion of radius/ulna and (in some cases) of tibia/fibula characteristic of the later stages (28–35) of talpid3 limb development. This evidence supports the hypothesis that cell death in the opaque patch plays a morphogenetic role in separation of radius/ulna and tibia/fibula. The digital plate of stage 32 (7½ days) normal limbs is characterized by massive necrosis of the interdigital tissue. In talpid3 forelimbs of stages 30–35 interdigital necrosis is absent, and there is no regression of the tissue between the digits (‘soft tissue syndactyly’). In talpid3 hindlimbs of stage 30–35 interdigital necrosis is either absent or much reduced, and there is little or no erosion of the soft tissue between the digits. This evidence supports the hypothesis that the morphogenetic role of interdigital cell death is in causing separation of the digits through shaping and remodelling the contours of the digital plate.

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Faculty Opinions recommendation of Vascular patterning regulates interdigital cell death by a ROS-mediated mechanism.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725326324.793505924 ◽

2015 ◽

Author(s):

Mark Lewandoski

Keyword(s):

Cell Death ◽

Vascular Patterning ◽

Interdigital Cell Death

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Interdigital cell death during limb development of the turtle and lizard with an interpretation of evolutionary significance

Development ◽

10.1242/dev.40.1.285 ◽

1977 ◽

Vol 40 (1) ◽

pp. 285-289

Author(s):

John F. Fallon ◽

Jo Ann Cameron

Keyword(s):

Cell Death ◽

Limb Development ◽

Evolutionary Significance ◽

Painted Turtles ◽

Interdigital Cell Death

Cell death accompanies the formation of free digits in birds and mammals. However, in species with webbing between the adult digits, little or no cell death occurs in the prospectively webbed region of the developing interdigit. Cell death does not occur during the formation of free digits in amphibians. In this paper we report that cell death accompanies the formation of the digits in snapping and painted turtles and in the skink (a lizard). We conclude that cell death accompanying the formation of free digits had its origin at the point of amniote emergence during evolution.

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Comparative proteomic analysis identifies protein disulfide isomerase and peroxiredoxin 1 as new players involved in embryonic interdigital cell death

Developmental Dynamics ◽

10.1002/dvdy.20404 ◽

2005 ◽

Vol 233 (2) ◽

pp. 266-281 ◽

Cited By ~ 17

Author(s):

S.W. Shan ◽

M.K. Tang ◽

D.Q. Cai ◽

Y.L. Chui ◽

P.H. Chow ◽

...

Keyword(s):

Cell Death ◽

Proteomic Analysis ◽

Protein Disulfide Isomerase ◽

Disulfide Isomerase ◽

Peroxiredoxin 1 ◽

Comparative Proteomic Analysis ◽

Interdigital Cell Death ◽

Protein Disulfide

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Formation of extra-digits induced by surgical removal of the apical ectodermal ridge of the chick embryo leg bud in the stages previous to the onset of interdigital cell death

Anatomy and Embryology ◽

10.1007/bf00310193 ◽

1987 ◽

Vol 176 (3) ◽

pp. 393-399 ◽

Cited By ~ 32

Author(s):

J. M. Hurle ◽

Y. Ganan

Keyword(s):

Cell Death ◽

Chick Embryo ◽

Apical Ectodermal Ridge ◽

Surgical Removal ◽

Interdigital Cell Death

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Expression of intermediate filament proteins during development of Xenopus laevis. I. cDNA clones encoding different forms of vimentin

Development ◽

10.1242/dev.105.2.279 ◽

1989 ◽

Vol 105 (2) ◽

pp. 279-298

Author(s):

H. Herrmann ◽

B. Fouquet ◽

W.W. Franke

Keyword(s):

Amino Acid ◽

Xenopus Laevis ◽

Intermediate Filament ◽

De Novo ◽

Mesenchymal Cell ◽

Amino Acid Sequences ◽

Cytoskeletal Proteins ◽

Cdna Clones ◽

Mammalian Development ◽

Intermediate Filament Proteins

To provide a basis for studies of the expression of genes encoding the diverse kinds of intermediate-filament (IF) proteins during embryogenesis of Xenopus laevis we have isolated and characterized IF protein cDNA clones. Here we report the identification of two types of Xenopus vimentin, Vim1 and Vim4, with their complete amino acid sequences as deduced from the cloned cDNAs, both of which are expressed during early embryogenesis. In addition, we have obtained two further vimentin cDNAs (Vim2 and 3) which are sequence variants of closely related Vim1. The high evolutionary conservation of the amino acid sequences (Vim1: 458 residues; Mr approximately 52,800; Vim4: 463 residues; Mr approximately 53,500) to avian and mammalian vimentin and, to a lesser degree, to desmin from the same and higher vertebrate species, is emphasized, including conserved oligopeptide motifs in their head domains. Using these cDNAs in RNA blot and ribonuclease protection assays of various embryonic stages, we observed a dramatic increase of vimentin RNA at stage 14, in agreement with immunocytochemical results obtained with antibody VIM-3B4. The significance of very weak mRNA signals detected in earlier stages is discussed in relation to negative immunocytochemical results obtained in these stages. The first appearance of vimentin has been localized to a distinct mesenchymal cell layer underlying the neural plate or tube, respectively. The results are discussed in relation to programs of de novo synthesis of other cytoskeletal proteins in amphibian and mammalian development.

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Cell death and the development of limb form and skeletal pattern in normal and wingless (ws) chick embryos

Development ◽

10.1242/dev.30.3.753 ◽

1973 ◽

Vol 30 (3) ◽

pp. 753-772

Author(s):

J. R. Hinchliffe ◽

D. A. Ede

Keyword(s):

Cell Death ◽

Phenotypic Expression ◽

Mesenchymal Cell ◽

Limb Bud ◽

Death Process ◽

Electron Microscopic ◽

Wide Range ◽

Skeletal Pattern ◽

Autoradiographic Analysis ◽

Mutant Phenotypes

The wingless condition resulting from the action of the sex-linked wingless (ws) gene arises from the precocious appearance of cell death in the anterior necrotic zone (ANZ) of the forelimb-bud at stage 19 (3 days) and its progressive extension beyond its normal area during stages 20–23. A similar though less pronounced effect occurs in the hindlimb-bud. Although some wingless hindlimb-buds are normal, others are affected by the precocious appearance of cell death in the ANZ. The ws wingless mutant resembles the different wingless mutant investigated by Zwilling (1956) in that the apical ectodermal ridge (AER) is absent in most ws wing-buds. AER absence could be due to ws mesenchymal cell death interfering with the production of apical ectodermal maintenance factor (AEMF), which Zwilling claims is necessary to maintain the AER which plays an essential role in inducing limb outgrowth. Wingless mutant phenotypes range from birds with rudimentary wings and normal legs through a modal type with forelimbs absent and hindlimbs normal to wingless and legless forms showing a high degree of expressivity. Individual wingless embryos vary in the degree to which the precocious ANZ appearing at 3 days is extended into the limb-bud and the wide range of wingless phenotypic expression is attributed to this variation. Electron microscopic and histochemical analysis of the cell death process in wingless wing-buds revealed the presence of both isolated dead cells and macrophages, which contained intense acid phosphatase activity. These findings are interpreted as showing that isolated dead cells are ingested by neighbouring mesenchymal cells which thus become transformed into macrophages, first ingesting and then digesting further dead cells. A study was made of the origin of the anomalous hindlimb condition, including absence or reduction of the tibia and digits, characteristic of severely affected wingless embryos. Autoradiographic analysis of the pattern of 35SO4 uptake revealed that at stage 24/5 (4½ days) wingless hindlimb-buds which were smaller than normal had a normal prospective fibula region, but that the prospective tibia region was small or absent. Thus the effect of a precocious hindlimb ANZ at stages 19–22 is to reduce or delete the pre-axial prospective tibia at stage 24/5.

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