Biomedical subjects

Y Gañan

Publications and source records attributed to Y Gañan.

25 records · Page 2Linked to original sources

PubMed1993

Internucleosomal DNA fragmentation and programmed cell death (apoptosis) in the interdigital tissue of the embryonic chick leg bud.

In this work we have attempted to characterize the programmed cell death process in the chick embryonic interdigital tissue. Interdigital cell death is a prominent phenomenon during limb development and has the role of sculpturing the digits. Morphological changes in the regressing interdigital tissue studied by light, transmission and scanning electron microscopy were correlated with the occurrence of internucleosomal DNA fragmentation, evaluated using agarose gels. Programming of the cell death process was also analyzed by testing the chondrogenic potential of the interdigital mesenchyme, in high density cultures. Our results reveal a progressive loss of the chondrogenic potential of the interdigital mesenchyme, detectable 36 hours before the onset of the degenerative process. Internucleosomal DNA fragmentation was only detected concomitant with the appearance of cells dying with the morphology of apoptosis, but unspecific DNA fragmentation was also present at the same time. This unspecific DNA fragmentation was explained by a precocious activation of the phagocytic removal of the dying cells, confirmed in the tissue sections. From our observations it is suggested that programming of cell death involves changes before endonuclease activation. Further, cell surface changes involved in the phagocytic uptake of the dying cells appear to be as precocious as endonuclease activation.

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PubMed1993

In vivo experimental induction of interdigital tissue chondrogenesis in the avian limb bud results in the formation of extradigits. Effects of local microinjection of staurosporine, zinc chloride and growth factors.

In previous studies we had found that at late stages of development, when the early patterning control mechanism have ceased to act, the chick limb bud is able to form fully differentiated extradigits by subjecting the interdigital spaces to ectoderm removal. In this study we attempted to mimic this phenomenon by using local microinjections of substances which presumably have a biological action on the interdigital mesenchyme. Microinjection of staurosporine results in the formation of fully differentiated extradigits. The action of this drug appears to be due to the induction of chondrogenesis after the inhibition of the protein kinase C. Zinc chloride administration also causes ectopic chondrogenesis but it seems to act by arresting the interdigital cell death program through endonuclease inhibition. A clear differentiation of the zinc-induced cartilages into extradigits was no detected. This can be explained by the accompanying damage caused by zinc in the growing limb mesenchyme as deduced by the high incidence of hypophalangy in the normal digits. Both TGF beta 1 and TGF beta 2 have a weak effect as inducers of interdigital chondrogenesis; presumably they act by inducing chondrogenetic differentiation. Neither FGF nor EGF has any effect when administered by local microinjection. These results show that ectopic interdigital chondrogenesis induced by drug administration results in the differentiation of extradigits. This suggests that once a cartilage is formed in the autopodium it triggers a new signalling stage which leads to the morphogenesis of a digit. This morphogenetic process involves the patterning of skeleton, joints and tendons. In accordance with these observations, it can be proposed that early patterning of the limb results in the establishment of an autopodium with a defined but still plastic skeletal distribution pattern, while morphogenesis of each autopodial element would take place at a second stage by the activation of new signalling processes.

Alkaloids↗

PubMed1992

Interdigital chondrogenesis and extra digit formation in the duck leg bud subjected to local ectoderm removal.

In the chick embryo the interdigital tissue in the stages previous to cell death exhibits in vitro a high chondrogenic potential, and forms extra digits when subjected in vivo to local ectodermal removal. In the present work we have analyzed the chondrogenic potential both in vivo and in vitro of the interdigital mesenchyme of the duck leg bud. As distinct from the chick, the interdigital mesenchyme of the duck leg bud exhibits a low degree of degeneration, resulting in the formation of webbed digits. Our results show that duck interdigital mesenchyme exhibits also a high chondrogenic potential in vitro until the stages in which cell death starts. Once cell death is finished chondrogenesis becomes negative and the interdigital mesenchyme forms a fibroblastic tissue. In vivo the interdigital mesenchyme of the duck leg bud subjected to ectoderm removal forms ectopic foci of chondrogenesis with a range of incidence similar to that in the chick. Unlike those of the chick the ectopic cartilages of the duck are rounded and smaller, and appear to be located at the distal margin of the interdigital mesenchyme. Formation of extra digits in the duck occurs with a lower incidence than in the chick. It is concluded that ectopic chondrogenesis and formation of extra digits is related to the intensity of interdigital cell death. The non-degenerating interdigital mesenchymal cells destined to form the interdigital webs of the duck appear to contribute very little to the formation of interdigital cartilages.

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PubMed1990

Experimental analysis of the role of ECM in the patterning of the distal tendons of the developing limb bud.

We have shown previously that from stage 27 the distal growing region of the limb exhibits a tenascin-rich sheet of extracellular matrix termed the "mesenchyme lamina" (ML), which runs from the ectodermal basement membrane in a proximal direction until it contacts the distal tip of the muscle blocks. This study reports experimental evidence that the mesenchyme lamina is a pretendinous structure that controls the spatial organization of the flexor and extensor tendons of the distal part of the chick leg. Two sets of experiments were designed to alter the ML position and examine subsequent tendon pattern formation. In a first series of experiments limbs with digits lacking phalangeal elements were induced by AER removal at stages 26 and 27. This procedure induced an abnormal arrangement of the ML around the distal tip of each terminal phalange of the truncated digit, which was followed by the development of a precisely similar pattern of abnormal extensor and flexor tendons. In the second set of experiments, an extradigit was induced to form in the interdigital mesenchyme through surgical removal of the marginal ectoderm of the third interdigit of stage 29 leg buds. By day 4 post-operation, a chondrogenic extradigit had formed, together with a ML that ran from the cartilage to the normal ventral flexor and dorsal extensor tendons. By day 6 post-operation, the experimentally induced ML had transformed into a tendinous structure connecting with the adjacent normal tendon. Both experiments show that the position of the ML defines the position of subsequent tendon development, thus supporting its role as a pretendinous structure which might be responsible for the alignment of the pretendinous condensing cells.

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PubMed1989

Experimental analysis of the in vivo chondrogenic potential of the interdigital mesenchyme of the chick leg bud subjected to local ectodermal removal.

Current in vitro investigations suggest that ectoderm plays a major role in limb morphogenesis by producing a diffusible factor which inhibits the chondrogenesis of the underlying mesenchyme. In the present work we report evidence supporting such an ectodermal role in vivo. Surgical removal of the marginal ectoderm from the third interdigit of chick leg buds at stages 27 to 30 induces the formation of PNA-positive prechondrogenic mesenchymal condensations 15 hr after the operation. The incidence of prechondrogenic condensations achieved 47, 95.2, and 92.8 of the experimental embryos of stages 27, 28, and 29, respectively. This high rate of prechondrogenic aggregate formation contrasted with a lower incidence of ectopic cartilage formation detectable by Alcian blue staining 40 hr after the operation. The sequential analysis of the experimental interdigits by means of peanut lectin labeling suggests that a number of prechondrogenic condensations undergo disaggregation 20 and 30 hr after the operation failing to form fully differentiated cartilages. When ectoderm removal was accompanied by the elimination of a variable amount of interdigital mesenchyme the incidence of prechondrogenic aggregates showed little differences but the formation of fully differentiated cartilages was reduced at a rate proportional to the amount of interdigital mesenchyme removed. From this study it can be concluded that the ectoderm in vivo appears to inhibit the process of aggregation of the mesenchymal cells to form prechondrogenic condensations. Furthermore our results suggest that as observed in vitro (C. P. Cotrill, C. Archer, and L. Wolpert, 1987, Dev. Biol. 122, 503-515) the transformation of prechondrogenic aggregates into fully differentiated cartilage requires the involvement of a critical amount of mesenchymal cells.

Alcian Blue↗

PubMed1987

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.

In the present work we have studied the mechanism of formation and the possible morphogenetic significance of the process of ectopic chondrogenesis induced by surgical removal of AER of the interdigital spaces of the chick leg bud at stage 28-30 (Hurle and Gañan 1986). Our results show that ridge removal causes condensation and rounding of the underlaying mesenchymal cells followed by chondrogensis. The long-term study of the fate of these ectopic cartilages shows that in a high percentage of the cases the cartilages undergo morphogenesis taking by day 10 of incubation the appearance of the two distal phalanges of an extra-digit. These extra-digits lack tendons and are joined by thin interdigital membranes to the neighboring digits.

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PubMed1986

Interdigital tissue chondrogenesis induced by surgical removal of the ectoderm in the embryonic chick leg bud.

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: removal of the AER of the interdigit; removal of the dorsal ectoderm of the interdigit. After the operation embryos were sacrificed at 10-12 h 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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