Coordinated and sequential activation of neutral and acidic DNases during interdigital cell death in the embryonic limb.

Interdigital tissue regression during embryonic development is one of the most representative model systems of morphogenetic cell death, but the degenerative cascade accounting for this process awaits clarification. Although the canonical apoptotic caspase pathway appears to be activated in the interdigital mesenchyme committed to die, neither genetic nor chemical blockage of caspases or their downstream effectors, is sufficient to prevent cell death. Hence, alternative and/or complementary dying pathways must also be responsible for this degenerative process. In this work we have chosen to study the endonucleases during the regression of the interdigital tissue of avian embryos to gain insights into the molecular mechanisms accounting for programmed cell death in this system. We show that caspase activated DNase, which is a neutral DNase associated with the caspase apoptotic pathway, appears to be the main endonuclease only at an initial phase of interdigit regression. However at peak stages of the degenerative process, the acidic DNases L-DNase II and lysosomal DNase IIB become predominant in the system and markers for cell autophagy become moderately up-regulated. Consistent with the activation of acidic endonucleases we observed that microenvironmental pH value in the interdigits decreased to levels only appropriate for acidic enzymes. Furthermore, we found that overexpression of lysosomal DNase IIB in embryonic limb mesoderm promoted cell death, which was also accompanied by up-regulation and activation of L-DNase II. Up-regulation of acidic DNases was maintained in interdigits explanted to culture dishes, where the participation of exogenous professional phagocytes of hematopoietic origin is avoided. Finally, and consistent with all our findings, up-regulation of acidic DNases was much reduced in the webbed interdigits of duck embryos, characterized by a rudimentary interdigital degenerative process. We conclude that the regression of the interdigital tissue involves a coordinated and sequential activation of the caspase and lysosomal degenerative molecular cascades.

Four and a half domain 2 (FHL2) scaffolding protein is a marker of connective tissues of developing digits and regulates fibrogenic differentiation of limb mesodermal progenitors.

Four and a half LIM domain 2 (FHL2) is a multifunctional scaffolding protein of well-known function regulating cell signalling cascades and gene transcription in cancer tissues. However, its function in embryonic systems is poorly characterized. Here, we show that Fhl2 is involved in the differentiation of connective tissues of developing limb autopod. We show that Fhl2 exhibits spatially restricted and temporally dynamic expression around the tendons of developing digits, interphalangeal joint capsules, and fibrous peridigital tissue. Immunolabelling analysis of the skeletal progenitors identified a predominant, but not exclusive, cytoplasmic distribution of FHL2 being associated with focal adhesions and actin cytoskeleton. In the course of chondrogenic differentiation of cultures of limb skeletal progenitors, the expression of Fhl2 is down-regulated. Furthermore, cultures of skeletal progenitors overexpressing Fhl2 take on a predominant fibrogenic appearance. Both gain-of-function and loss-of-function experiments in the micromass culture assays revealed a positive transcriptional influence of Fhl2 in the expression of fibrogenic markers including Scleraxis, Tenomodulin, Tenascin C, ßig-h3, and Tgif1. We further show that the expression of Fhl2 is positively regulated by profibrogenic signals including Tgfß2, all-trans-retinoic acid, and canonical Wnt signalling molecules and negatively regulated by prochondrogenic factors of the bone morphogenetic protein family. Expression of Fhl2 is also regulated negatively in immobilized limbs, but this influence appears to be mediated by other connective tissue markers, such as Tgfßs and Scleraxis.

Expression of Sox8, Sox9 and Sox10 in the developing valves and autonomic nerves of the embryonic heart.

We describe the expression pattern of Sox8, Sox9 and Sox10 during the development of the chick embryo heart. These Sox genes constitute the group E of the large Sox family of transcription factors. We show that the expression of Sox8, Sox9 and Sox10 in the developing heart correlates with heart septation and with the differentiation of the connective tissue of the valve leaflets. Sox10 appears also as a specific marker of developing heart nerves. These findings fit with the occurrence of morphological and functional anomalies of the heart reported in humans deficient for Sox9 and Sox10.

Morphologic characteristics and structure of surface excrescences (Lambl's excrescences) in the normal aortic valve.

The incidence, morphologic characteristics and structure of surface valve excrescences (Lambl's excrescences) were studied by transmission and scanning electron microscopy of the aortic valve of 56 human subjects, age range birth to 91 years, without cardiac disease. Valve excrescences consisting of a core of connective tissue covered by the endocardium were observed in 90% of the subjects; the incidence was significantly lower in patients in the first decade of life. Two types of excrescences, lamellar and filiform, were found. Lamellar excrescences are located along the lower boundary of the lunulas and occurred more often in those younger than 30 years. Filiform excrescences appear most often in the nodulus Arantius and in the free-margin of the cusps. The excrescences of the nodulus are the most numerous. Free-margin excrescences are the least numerous and occur more frequently in persons older than 40 years. The connective tissue core of the filiform excrescences contains abundant collagen fibrils and elastic material arranged in apposed layers with different collagen fibril orientation. A circular zone devoid of identifiable connective tissue is present at the center of the filiform excrescences.

Bone morphogenetic proteins regulate interdigital cell death in the avian embryo.

The embryonic limb bud provides an excellent model for analyzing the mechanisms that regulate programmed cell death during development. At the time of digit formation in the developing autopod, the undifferentiated distal mesodermal cells may undergo or chondrogenic differentiation or apoptosis depending whether they are incorporated into the future digital rays or into the interdigital spaces. Both chondrogenesis or apoptosis are induced by local BMPS. However, whereas the chondrogenic-promoting activity of BMPs appears to be regulated through the BMPR-1b receptor, the mechanism by which the BMPs execute the death program remains unknown. The BMP proapoptotic activity requires the expression of members of the msx family of closely related homeobox-containing genes and is finally mediated by caspase activation, but the nature of the caspase(s) directly responsible for the cell death is also unknown. Finally, other growth factors present in the developing autopod at the stages of digit formation such as members of the FGF and TGF beta families modulate the ability of BMPs to induce cell death or chondrogenesis.

Morphology and significance of programmed cell death in the developing limb bud of the vertebrate embryo.

Cell death constitutes a basic mechanism accounting for many morphogenetic and histogenetic events during normal and abnormal development of embryonic organs and tissues. This article focuses on the major areas of mesodermal cell death occurring during vertebrate limb development. In early stages of limb development, cell death appears to reduce the amount of mesodermal tissue destined to form the anlage of the autopodium. In later stages, cell death plays a role sculpturing the shape of the digits. The morphology of the dying cells corresponds with apoptosis, but internucleosomal DNA fragmentation by endonuclease activation does not appear to be a precocious feature. The cell death program can be inhibited in vivo and in vitro by changing the environmental conditions of the prospective dying cells up to 6-10 h before death. In this review, we survey possible factors controlling the establishment of the cell death program. Information concerning the biochemical basis of cell death in the developing limb is also revised. Finally, the possible role of genes whose pattern of expression is coincident with the dying processes is discussed.

Scanning and light microscope studies of the development of the chick embryo semilunar heart valves.

The development of the semilunar valves of the great arteries was studied by light and scanning electron microscopy in the chick embryo. The results show that three distinct developmental periods can be distinguished. The formation of the anlage of the valves takes place in the first period (stages 26--29). These early anlage consist of three pyramidal shaped cusps formed by a core of loosely packed mesenchymal cells covered by a flattened endothelium. In the second period (stages 30--35) the cusps undergo excavation on their distal face. Morphololgical evidence is reported suggesting that this excavation process is produced by an initial solid ingrowth of the endothelium of the arterial face of the cusps which is immediately luminated by detachment of cells towards the bloodstream and by cell death. The histogenesis of the valves takes place in the third period (from stage 36 until hatching). It was observed that during this period some myocardial cells of the outflow tracts of the ventricles invade the valvular tissue and that in the upper part of the cusps a prominent fibrous layer is formed.

Maturation of the extracellular material of the semilunar heart values in the mouse. A histochemical analysis of collagen and mucopolysaccharides.

The developmental changes of collagen and mucopolysaccharides in the semilunar values of the mouse were studied during the embryonic, fetal and postnatal period. The valvular collagen was investigated using Van Gieson and Sirius red-polarization microscopy methods. These procedures showed that the establishment of the fibrosa layer of the cusps does not occur until the second week of the postnatal period. The nature and distribution of the valvular mucopolysaccharides were investigated by staining with Alcian blue at specific pH values and at various critical electrolyte concentrations, with the appropriate enzymatic controls using Streptomyces and testicular hyaluronidase. The results show that hyaluronate and chondroitin 4- and 6-sulphate are the major components during the embryonic and fetal period. In the older fetal stages and during the postnatal period the relative amount of hyaluronate decreases, while chondroitin sulphate increases. It is concluded from this study that the maturation of the valves occurs over a long period of the postnatal life.

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.

Extracellular matrix modifications in the interdigital spaces of the chick embryo leg bud during the formation of ectopic digits.

In previous studies we have observed that the interdigital mesenchyme of the chick leg bud, in the stages preceding the onset of cell death, retains a significant regulatory potential, forming ectopic extra digits under a variety of surgical manipulations. Most evidence suggests that interdigital extra digits are caused by the abolition of local antichondrogenic effects operating in the interdigital spaces under normal conditions rather than by modifications of the signalling mechanisms accounting for the normal patterning of the digits in early stages of development. The interdigital spaces exhibit a complex scaffold of extracellular matrix with well-defined domains of spatial distribution of type I and type VI collagens, tenascin, fibronectin, laminin and elastic matrix components that have been proposed to play a role in the establishment of the non-chondrogenic fate of the interdigital tissue in situ. In an attempt to analyze this possible role of the interdigital extracellular matrix (ECM), in the present work we have studied changes in the pattern of ECM distribution associated with the formation of extra digits. Extra digits were induced by making a T-cut in the third interdigital space of the leg but of stage 29 HH chick embryos. Subsequent modifications of the ECM were detected immunohistochemically in whole-mount specimens using laser confocal microscopy. Our results reveal that in the first hours after the operation, changes in the ECM apparently related to the healing of the wound cause a significant reorganization of the normal ECM scaffold of the interdigit. In addition, chondrogenesis of the interdigital tissue is preceded by disappearance of elastin fibers in the interdigital mesenchyme subjacent to the wound and by an intense deposition of tenascin. Tenascin deposition and loss of the elastin fibrillar scaffold were also observed preceding chondrogenesis in fragments of interdigital tissue explanted to culture conditions. The significance of these observations in relation to the establishment of the skeletal elements of the autopodium is discussed.

Cell shape and cytoskeletal organization of the endothelial cells of the semilunar heart valves in the developing chick.

The composition and arrangement of the cytoskeletal elements of the endothelium of the semilunar valves have been studied in the embryonic chick heart during the stages 30 to 38. Microtubules, vimentin intermediate filaments and actin microfilaments were constant components of the valvular endothelial cells in the studied stages. Scanning electron microscopy after Triton-X-100 extraction revealed significant differences in the tridimensional arrangement of the cytoskeleton in the course of valve development. In the ventricular face of the cusps the cytoskeletal elements displayed a progressive longitudinal alignment, while in the arterial face of the cusps the cytoskeleton maintained the appearance of a network. Transmission electron microscopy revealed that these differences were especially prominent for vimentin intermediate filaments, although a similar tendency was also observed for microtubules. Microfilaments were scarce in the endothelial cells of both faces of the cusps, and the stress fibers typical of the endothelial cells of the adult valves were not observed in the embryonic material. The significance of these results in valve morphogenesis and histogenesis and a possible linkage with differences in the local characteristics of the blood flow are discussed.

Changes in the endothelial morphology of the developing semilunar heart valves. A TEM and SEM study in the chick.

In view of recent evidence showing that shape and orientation of endothelial cells is determined by blood flow, the endothelium of the semilunar valves was studied in the developing chick heart using transmission and scanning electron microscopy. The results reveal significant developmental modifications of endothelial morphology and structure. These modifications can be linked to modifications of local blood flow and can also explain several aspects of valvular morphogenesis. The results substantially support the hypothesis of an involvement of hemodynamics in the development of the semilunar valves.

Malformations of the semilunar valves produced in chick embryos by mechanical interference with cardiogenesis. An experimental approach to the role of hemodynamics in valvular development.

The purpose of the present work was to analyze the role of hemodynamics in the morphogenesis and histogenesis of the semilunar valves. To achieve this goal we have studied the development of the chick semilunar valves in conditions of abnormal local flow. To obtain an abnormal pattern of local flow we have induced alterations of the cardiac septation process by mechanical interference of the development of the conus cordis. The malformations obtained by this procedure consisted of a spectrum of alterations in the process of incorporation of the aortic conus into the left ventricle. These malformations ranged from a simple widening of the outflow tract of the left ventricle to severe forms of double-outlet right ventricle and ventricular septal defects. Malformations of the semilunar valves consisting of extensive thickening of the leaflets and lack of maturation of the valve tissues were very often present in the malformed hearts. The malformation of the valve leaflets was more frequent and severe in the aortic valve at more advanced stages of development and in the hearts showing more severe alteration of the septation process. The absence of alterations in the semilunar valves of the control embryos and in the experimental embryos without alteration of the cardiac septation suggest a close relationship between the semilunar valves anomalies and the hemodynamic alterations present in the malformed hearts.

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.

Development of mouse semilunar valves.

The development of the semilunar valves of the great arteries of the mouse is studied by light and scanning electron microscopy. The earliest anlage of the valves is observed at day 11.5 of gestation, as three pairs of tubercles protruding towards the lumen of the truncus arteriosus. These early cusps consist of a core of mesenchymal tissue covered by the endocardium. From day 12.5 to 14 a process of excavation takes place in the arterial face of the cusps resulting in the achievement of their final shape. Our observations suggest that the excavation process is produced by a selective growth of the free edges of the cusps. Numerous pieces of evidence are reported suggesting that the selective growth of the cusps is due to an inductive interaction between the endocardium of the arterial face of the cusps and the underlying mesenchyme. The histogenesis of the cusps takes place very late in development and possibly continues into the postnatal period. The results are compared with studies made in other vertebrates.

Modification of the phalangeal pattern of the digits in the chick embryo leg bud by local microinjection of RA, staurosporin and TGF beta's.

Many experimental studies show that in the avian chick limb the digits are specified at early stages of development by characteristic concentrations within the limb mesoderm of a still unidentified morphogen diffusing from the posterior margin of the bud, linked with a specific pattern of homeobox gene expression. In all these studies, digits are distinguished by their size, morphology and phalangeal pattern rather than by their position within the autopodium. In this work we report the induction of digits that have otherwise normal morphology but lack an interphalangeal joint. This suggests that the patterning of these joints is not necessarily linked to the control of the outgrowth of the digital rays. Missing interphalangeal joints were induced by microinjection into the third interdigital space of the leg bud of stage 28 to 31 chick embryos of retinoic Acid (RA), staurosporine and TGF beta 1 and beta 2, but not by microinjection of FGF or EGF. Our results also suggest that the pattern of insertion of the long tendons and the formation of the flexor cutaneous pad at the plantar surface of the digits are both linked to the establishment of the interphalangeal joints.

The surface anatomy of the human aortic valve as revealed by scanning electron microscopy.

The anatomy of the human aortic valve was studied by SEM in 36 subjects without cardiac pathology who had died of various accidental causes. Villous and lamellar tissue excrescences were observed at the node of Arantius and at the limit between the lunules and the load-bearing portion of the leaflets. The morphology of these structures suggests that they represent areas in which valve tissue becomes detached into the bloodstream. Fenestrations were present in the lunules of 14 specimens, with a higher incidence in specimens from subjects who were middle-aged or older. Our observations suggest that fenestrations appear initially as small perforations which then coalesce to form larger apertures. Two main types of endothelial cells, elongated and polygonal were detected on the endothelial surface of the leaflets. Both types of cells display a constant mode of arrangement on the different segments of the leaflets (lunules, node of Arantius and load-bearing portion of the leaflet). The possible relationships between endothelial cell morphology and the pattern of mechanical stress to which the leaflets are subjected is discussed.

In vivo inhibition of programmed cell death by local administration of FGF-2 and FGF-4 in the interdigital areas of the embryonic chick leg bud.

The formation of the digits in amniote vertebrates is accompanied by a massive degeneration process that accounts for the disappearance of the interdigital mesenchyme. The establishment of these areas of interdigital cell death (INZs) is concomitant with the flattening of the apical ectodermal ridge (AER), but a possible causal relationship between these processes has not been demonstrated. Recent studies have shown that the function of the AER can be substituted for by implantation of beads bearing either FGF-2 or FGF-4 into the apical mesoderm of the early limb bud. According to these observations, if the onset of INZs is triggered by the cessation of the AER function, local administration of FGFs to the interdigital tissue prior to cell death should delay or inhibit interdigit degeneration. In the present study we have confirmed this prediction. Implanting Affi-gel blue or heparin beads pre-absorbed with either FGF-2 or FGF-4 into the interdigital tissue of the chick leg bud in the stages prior to cell death stimulates cell proliferation and causes the formation of webbed digits. Vital staining with neutral red confirmed an intense temporal inhibition of interdigital cell death after FGF treatment. This inhibition of interdigital cell death was not accompanied by modifications in the pattern of expression of Msx-1 or Msx-2 genes, which in normal development display a domain of expression in the interdigital tissue preceding the onset of degeneration.

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.