Control of skeletogenesis and programmed cell death in the developing avian limb bud by growth factors.
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Animals↗
Biomedical subjects
J M Hurle
Publications and source records attributed to J M Hurle.
Cardiac malformations induced after "in ovo" administration of OP-1 to the outflow tract in the developing chick.
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Animals↗
Morphological changes in the normal pattern of ventricular myoarchitecture in the developing human heart.
BACKGROUND: The aim of the present study was to describe the morphological changes in the normal pattern of ventricular myoarchitecture in the prenatal and adult human heart, to understand the three-dimensional organization of the muscle fibers and their active functional role in valvular dynamics. METHODS: We used dissection and histological techniques in 56 human hearts from fetuses and adults of both sexes. RESULTS: In all hearts, the ventricular wall was arranged in three different layers: superficial (subepicardial), middle, and deep (subendocardial) myocardium. The superficial and deep layers are present in both ventricles, whereas the middle layer is found only in the left ventricle. Age-related differences were noted in the pattern of myoarchitecture of the superficial layer, mainly in the fetal period, and especially in the right ventricle; however, the middle layer always shows a circumferential pattern, which is specially evident in elderly hearts. The ventricular fibers in the superficial and deep layers are anchored in the ventricular orifices. CONCLUSIONS: Our findings reveal that muscle fiber architecture showed age- but not sex-related differences. These variations may reflect a mechanism of adaptation of the heart to functional demands throughout life.
Adolescent↗
Immunohistological and ultrastructural study of the developing tendons of the avian foot.
The aim of the present report is to provide a detailed description of the morphogenesis and initial differentiation of the long tendons of the chick foot, the long autopodial tendons (LAT), from day 6 to day 11 of development. The fine structure of the developing LAT was studied by light and transmission electron microscopy. The characterization by immunofluorescent techniques of the extracellular matrix was performed using laser scanning confocal (tenascin, elastin, fibrillin, emilin, collagen type I, II, III, IV and VI) or routine fluorescence (tenascin, 13F4) microscopy. In addition, cell proliferation in pretendinous blastemas was analyzed by the detection of BrdU incorporation by immunofluorescence. The light microscopic analysis permitted the identification of different stages during LAT morphogenesis. The first stage is the formation of a thick ectoderm-mesenchyme interface along the digital rays, followed by the differentiation of the "mesenchyme lamina", an extracellular matrix tendon precursor, and ending with the formation and differentiation of the cellular condensation that forms the tendon blastema around this lamina. The immunofluorescence study revealed the presence and arrangement of the different molecules analyzed. Tenascin and collagen type VI are precocious markers of the developing tendons and remain present during the whole process of tendon formation. Collagen type I becomes mainly restricted to the developing tendons from day 7.5. Collagens type II and IV are never detected in the developing tendons, while a faint labeling for collagen type III is first detected at day 7. The analysis of the distribution of the elastic matrix components in the developing tendons is a major contribution of our study. Elastin was detected in the periphery of the tendons from day 8 and also in fibrils anchoring the tendons to the skeletal elements. At the same stage, emilin strongly stains the core of the tendon rods, while fibrillin is detected a little later. Our study indicates the existence of an ectoderm-mesoderm interaction at the first stage of the tendon formation. In addition, our results show the different spatial and temporal pattern of distribution of extracellular matrix molecules in developing tendons. Of special importance are the findings concerning the tendinous elastic matrix and its possible role in tendon maturation and stabilization.
Animals↗
Morphological analysis of the fish heart ventricle: myocardial and connective tissue architecture in teleost species.
Light and scanning electron microscopy were used to study the structure of the heart ventricle in three species of marine teleost fishes: the hake (Merluccius merluccius), the angler fish (Lophius piscatorius) and the sea bream (Pagellus centrodontus). Our findings show the ventricle to be shaped differently in each species: tubular in the hake, saccular in the angler fish and pyramidal in the sea bream. From a structural viewpoint, interest was centered on two aspects: organization of the myocardial fibres and arrangement of connective tissue. In hake and angler fish ventricles, the myocardium was exclusively trabecular in nature, whereas the bream ventricle, in addition to trabecular myocardium, presented a thin compact layer. Muscle fibres showed precise patterns of organization at the level of the ventricular orifices. With the techniques used the intramyocardial connective tissue was detected in the following ventricular zones: i) at the level of subepicardial and subendocardial spaces, ii) surrounding the myocardial fascicles, and iii) surrounding individual myocardial cells. According to this structural study, the pyramidal ventricle of the fish should be considered as a ventricular pump with greater efficiency.
Animals↗
Cell death in the embryonic developing limb.
In amniote vertebrates, the development of form and structure of the limb bud is accompanied by precise patterns of massive mesodermal cell death with morphological features of apoptosis. These areas of cell death appear to eliminate undifferentiated cells which are required only for a limited time period of limb development. Predictable skeletal and morphological anomalies of the limb occur when the pattern of cell death is modified in mutant species or under experimental conditions. Most evidence points to the occurrence of local triggering mechanisms to account for the establishment of the areas of cell death and the subsequent activation of cell death genes. Modifications of the extracellular matrix and diminution in the contribution of growth factors by neighbouring tissues appear as the most likely potential candidates for triggering the cell death program. Information on the genetical basis of cell death in the developing limb is very scarce. Among the increasing number of cell death genes identified in other cell death systems, such as p-53 and the ced-3/ICE and ced-9/ bcl-2 gene families, only bcl-2 has been studied in detail during limb development and yet, the information obtained is contradictory. Bcl-2 is not expressed in the areas of cell death of the developing limb, but normal limbs develop in mice with disruption of the bcl-2 gene. Obviously, the clarification of the role of the cell death genes constitute a major task in future studies of cell death in the developing limb.
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Pattern regulation in the chick autopodium at advanced stages of embryonic development.
In previous studies we have observed that the interdigital tissue of the chick embryo leg bud during the stages previous to interdigital cell death exhibits a considerable chondrogenic potentiality both in vivo and in vitro. In the present investigation we have carried out a variety of experimental manipulations of the chick leg bud at stage 29 to discover possible mechanisms accounting for interdigital ectopic chondrogenesis and extradigit formation. Our results show that the interdigital tissue is capable of forming an extradigit when temporarily isolated microsurgically and regrafted in its original location and after deletion of one of the adjacent digital primordia, suggesting that developing phalangeal cartilages exercise an inhibitory effect on chondrification in adjoining tissues. Furthermore, and of greater importance, ablation of the primordium of a digit is followed by normal development of the definitive digit if the wound surfaces are suitably apposed. These results reveal a considerable regulatory potential in the autopodium at advanced stages of development.
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Elastic extracellular matrix of the embryonic chick heart: an immunohistological study using laser confocal microscopy.
The "elastic matrix" constitutes a specialized component of the extracellular matrix which confers resiliency to tissues and organs subjected to repeated deformations. The role of the elastic matrix in living organisms appears to be of key importance since diseases characterized by expression of defective inherited genes which encode components of the elastic matrix lead to premature death. While the elastic matrix of adult organs has received a great deal of attention, little is known about when it first appears in embryonic tissues or its possible role in developing organs. In the present study we have performed an immunohistochemical study of the distribution of elastin and three additional components often associated with elastic matrices in adult tissues (i.e., fibrillin, emilin, and type VI collagen) during the development of the chicken embryonic heart. The three-dimensional arrangement of these components was established through the observation of whole-amount specimens with scanning laser confocal microscopy. Our results revealed three different periods of heart development regarding the composition of the elastic matrix. Prior to stage 21 the embryonic heart lacks elastin but exhibits a matrix scaffold of fibrillin and emilin associated with the endocardium and the developing cardiac jelly. Between stages 22 and 29 the heart shows a transient elastic scaffold in the outflow tract which contains elastin, fibrillin, and emilin. Elastin-positive fibrillar material is also observed during these stages in the base of the atrioventricular cushion adjacent to the myocardial wall. In addition, emilin-positive material appears to be associated with the zones of formation of ventricular trabeculae. Collagen type VI was not detected during these early stages. From stage 30 to stage 40 a progressive modification of the pattern of distribution of elastin, fibrillin, emilin, and collagen type VI is observed in association with the formation of the definitive four-chambered heart. The distribution of the elastic scaffold in the outflow tract appears to be rearranged and becomes restricted to the roots of the main arteries. Each of the components studied here is also deposited at increasing levels in the developing valvular apparatus including the valve leaflets and the chordae tendinea. The components are also present in the subendocardial space where they form aligned fibrillar tracts, an arrangement suggestive of a role in ventricular contractile function. The epicardium constitutes an additional region of elastic matrix deposition during these later stages and contains elastic, fibrillin, and collagen type VI.(ABSTRACT TRUNCATED AT 400 WORDS)
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Formation of extra digits in the interdigital spaces of the chick leg bud is not preceded by changes in the expression of the Msx and Hoxd genes.
By in situ hybridization we studied the expression patterns of Msx and Hoxd genes during the late development of the chick leg autopodium (foot) and compared them to patterns during the experimental development of interdigital extra digits. Extra digits are induced in the third interdigital space after various experimental manipulations, such as transient isolation of the interdigit, or removal of the interdigital marginal ectoderm and mesoderm. Msx1 and Msx2 are normally expressed in the interdigital tissue programmed to die. Our experiment changes the fate of the interdigital tissue from cell death to chondrogenesis and provides a good model for studying Msx involvement in defining areas of programmed cell death. Among the proposed roles of Hoxd genes is their involvement in the specification of digit identity early in development. The induction of extra digits allows us to examine whether this new morphogenetic commitment of the interdigital tissues involves changes in the domains of expression of Hoxd genes. Our results show that extra digits develop without a previous modification of the normal pattern of expression of Msx or Hoxd genes. This observation does not support the correlation between the expression of Msx genes and programmed cell death and suggests a role for these genes in maintaining the interdigital tissue in an undifferentiated state. Our results show that an increased number of digits can be formed without modifications in the pattern of expression of the 5'-located Hoxd genes and suggest the existence of latent or residual digit organization mechanisms past the time when digits are normally determined, independent of Hoxd gene expression.
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Elastin exhibits a distinctive temporal and spatial pattern of distribution in the developing chick limb in association with the establishment of the cartilaginous skeleton.
In this work we have analyzed the presence of elastic components in the extracellular matrices of the developing chick leg bud. The distributions of elastin and fibrillin were studied immunohistochemically in whole-mount preparations using confocal laser microscopy. The association of these constituents of the elastic matrix with other components of the extracellular matrix was also studied, using several additional antibodies. Our results reveal the transient presence of an elastin-rich scaffold of extracellular matrix fibrillar material in association with the establishment of the cartilaginous skeleton of the leg bud. The scaffold consisted of elastin-positive fibers extending from the ectodermal surface of the limb to the central cartilage-forming regions and between adjacent cartilages. Fibrillin immunolabeling was negative in this fibrillar scaffold while other components of the extracellular matrix including: tenascin, laminin and collagens type I, type III and type VI; appeared codistributed with elastin in some regions of the scaffold. Progressive changes in the spatial pattern of distribution of the elastin-positive scaffold were detected in explant cultures in which one expects a modification in the mechanical stresses of the tissues related to growth. A scaffold of elastin comparable to that found in vivo was also observed in high-density micromass cultures of isolated limb mesodermal cells. In this case the elastic fibers are observed filling the spaces located between the cartilaginous nodules. The fibers become reoriented and attach to the ectodermal basal surface when an ectodermal fragment is located at the top of the growing micromass. Our results suggest that the formation of the cartilaginous skeleton of the limb involves the segregation of the undifferentiated limb mesenchyme into chondrogenic and elastogenic cell lineages. Further, a role for the elastic fiber scaffold in coordinating the size and the spatial location of the cartilaginous skeletal elements within the limb bud is also suggested from our observations.
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Age-dependent dystrophic calcification of the aortic valve leaflets in normal subjects.
The aortic valves of 43 subjects (14 females and 29 males, mean age 48.9 +/- 22.2 years, range 3-88 years), dying from accidental causes and without any previous record of cardiovascular disease, were studied. The whole aortic root was removed and morphological features recorded. Calcium content was determined by atomic absorption spectrophotometry. Calcium content sustained a positive significant correlation (p < 0.001) with age. Comparison of regression lines from subjects of different age groups disclosed a significantly greater slope in the regression line of persons older than 50 years than in those of the younger population (p < 0.05). The increase with age of calcium content displayed no significant differences between male and female nor between any of the three aortic cusps. The presence of Lambl's excrescences on the aortic leaflets became more frequent with age and was associated with a higher calcium content.
Adolescent↗
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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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↗
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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Structural arrangement of the extracellular matrix network during myocardial development in the chick embryo heart.
We analyzed the extracellular matrix and the connective tissue of the developing chick myocardium ("myocardial interstitium"). The importance of this myocardial element for heart function has been well documented both for the normal and pathologic adult hearts. However, little information is available on the organization of the embryonic myocardial interstitium and its modifications during development and increasing intracardiac pressure. In the present study we used light and scanning electron microscopic techniques, and lectin probes to study the interstitium of the ventricular myocardium of chick embryos from stage 29 (day 6 of development) until hatching. Our observations trace the progressive appearance and organization of the elements of the extracellular matrix, comprising the epimysium, perimysium and endomysium, which form a well-defined architectural network. Finally, we discuss the role of these elements of the extracellular matrix and their possible relation with the biomechanical properties of developing heart.
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Histochemical and ultrastructural changes in the extracellular matrix of the developing chick semilunar heart valves.
The present study analyzes the composition and organization of the extracellular matrix (ECM) and its changes in the course of development of the chick embryo semilunar heart valves. In the present work we have employed chick embryos from stage 29 until hatching, using silver and picrosirius red staining, lectin probes and light and transmission electron microscopy. Our results show that during semilunar valve development a series of elements arise and are organized in the ECM which seem to be more closely related to the maintenance of the structural and biomechanical properties of the valvular leaflets than with morphogenetic processes per se.
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Immunofluorescent localization of tenascin during the morphogenesis of the outflow tract of the chick embryo heart.
The cono-truncus constitutes a complex segment of the developing heart that gives rise to the outflow tract of the ventricles and root of the pulmonary and aortic arteries. Numerous studies have revealed that the extracellular matrix plays a relevant role in most morphogenetic processes modulating cell behaviour. By means of immunofluorescence, we studied the distribution and possible involvement of tenascin during morphogenesis of the conus and truncus in chick embryo hearts between days 4.5-10 of incubation. Tenascin is an extracellular matrix glycoprotein with a significant role in morphogenesis and cell and tissue differentiation. Our results reveal a specific distribution of tenascin in the areas of the cono-truncus undergoing significant structural changes during morphogenesis of this cardiac segment, appearing mainly in the mesenchymal layer subjacent to the myocardial layer, the cono-truncal ridges and the aorto-pulmonary septum. The distribution of tenascin was compared and contrasted with that of collagen type I, which constitutes a further component of the extracellular matrix common to most developing connective tissues.
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Histogenesis of the semilunar valves: an immunohistochemical analysis of tenascin and type-I collagen distribution in developing chick heart valves.
The development of the semilunar valves takes place in association with septation of the outflow tract in the embryonic heart. Although numerous studies have focused on this process, the causal mechanisms of valvular development remain obscure. This paper reports an immunohistochemical analysis of tenascin and type-I collagen distribution in developing chick heart valves. Tenascin is a glycoprotein that is present on some embryonic extracellular matrices. It plays several significant roles in tissue differentiation, cell growth, and tissue interactions; it is also important for the formation of specific zones of connective tissue that fulfill mechanical functions. Our results show that tenascin is present during valvular morphogenesis and histogenesis, and that its distribution is associated with zones specialized in bearing mechanical loads.
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