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Biomedical subjects

M A Ros

Publications and source records attributed to M A Ros.

At least 19 recordsLinked to original sources
PubMed1999

The recombinant limb as a model for the study of limb patterning, and its application to muscle development.

The recombinant limb is a model system that has proved fruitful for analyzing epithelial-mesenchymal interactions and understanding the functional properties of the components of the limb bud. Here we present an overview of some of the insights obtained through the use of this technique. Among these are the understanding that fore or hind limb identity is inherent to the limb bud mesoderm, that the apical ectodermal ridge (AER) is a permissive signaling center and that the limb bud ectoderm plays a central role in the control of dorsoventral polarity. Recombinant limb studies have also allowed the identification of the affected tissue component in several limb mutants. More recently this model has been applied to the study of regulation of gene expressions related to patterning. In this report we use recombinant limbs to analyze pattering of the Pax3 expressing limb muscle cell lineage in the early stages of limb development. In recombinant limbs made without the zone of polarizing activity (ZPA), myoblasts appear intermingled with other mesodermal cells at the beginning of the recombinant limb development. Rapidly thereafter, the muscle precursors segregate and organize around the central forming chondrogenic core of the recombinant. Although this segregation is reminiscent of that occurring during normal development, the myoblasts in the recombinant fail to proliferate appropriately and also fail to migrate distally. Consequently, the muscle pattern in the recombinant limb is defective indicating that normal patterning cues are absent. However, recombinant limbs polarized with a ZPA exhibited a larger mass of muscle cells and a more normal morphogenesis, supporting a role for this signaling center in limb muscle development. Finally, we have ruled out host somite contributions to recombinant limbs by grafting chick recombinant limbs to quail hosts. This initial report demonstrates the value of the recombinant limb model system for dissecting the environmental cues required for normal muscle limb patterning.

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PubMed1998

Pitx2 participates in the late phase of the pathway controlling left-right asymmetry.

Pitx2, a member of the bicoid-related family of homeobox-containing genes, is asymmetrically expressed in the left lateral plate mesoderm and derived tissues during chick and mouse development. Modifications of Pitx2 pattern of expression in the iv mouse mutation correlate with the situs alterations characteristic of the mutation. Misexpression experiments demonstrate that Shh and nodal positively regulate Pitx2 expression. Our results are compatible with a Pitx2 function in the late phase of the gene cascade controlling laterality.

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PubMed1998

The expression and regulation of chick EphA7 suggests roles in limb patterning and innervation.

Eph receptors and their ligands, the ephrins, have been implicated in early patterning and axon guidance in vertebrate embryos. Members of these families play pivotal roles in the formation of topographic maps in the central nervous system, the formation of brain commissures, and in the guidance of neural crest cells and motor axons through the anterior half of the somites. Here, we report a highly dynamic expression pattern of the chick EphA7 gene in the developing limb. Expression is detected in discrete domains of the dorsal mesenchyme from 3 days of incubation. The expressing cells are adjacent to the routes where axons grow to innervate the limb at several key points: the region of plexus formation, the bifurcation between dorsal and ventral fascicles, and the pathway followed by axons innervating the dorsal muscle mass. These results suggested a role for EphA7 in cell-cell contact-mediated signalling in dorsal limb patterning and/or axon guidance. We carried out experimental manipulations in the chick embryo wing bud to alter the dorsoventral patterning of the limb. The analyses of EphA7 expression and innervation in the operated wings indicate that a signal emanating from the dorsal ectoderm regulates EphA7 in such a way that, in its absence, the wing bud lacks EphA7 expression and shows innervation defects at the regions where the gene was downregulated. EphA7 downregulation in the dorsal mesenchyme after dorsal ectoderm removal is more rapid than that of Lmx-1, the gene known to mediate dorsalisation in response to the ectodermal signal. These results add a new gene to the dorsalisation signalling pathway in the limb. Moreover, they implicate the Eph receptor family in the patterning and innervation of the developing limb, extending its role in axon pathfinding to the distal periphery.

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PubMed1997

Limb initiation and development is normal in the absence of the mesonephros.

With rapid progress in understanding the genes that control limb development and patterning interest is becoming focused on the factors that permit the emergence of the limb bud. The current hypothesis is that FGF-8 from the mesonephros induces limb initiation. To test this, the inductive interaction between the Wolffian duct and intermediate mesoderm was blocked rostral to the limb field, preventing mesonephric differentiation while maintaining the integrity of the limb field. The experimental outcome was monitored by following expression of cSim1 and Lmx1, molecular markers for the duct and the mesonephros, respectively. Evidence is presented that the intermediate mesoderm undergoes apoptosis when the inductive interaction with the Wolffian duct is blocked. fgf-8 expression was undetectable in the mesonephric area of embryos with confirmed absence of mesonephros; nevertheless, limb buds formed and limb development was normal. The mesonephros in general, and specifically its fgf-8 expression, was shown to be unnecessary for limb initiation and development; the hypothesis linking the mesonephros and limb development is not supported. Further studies of axial influences on limb initiation should now concentrate on medial structures such as Hensen's node and paraxial mesoderm; the alternative that no axial influences are required should also be examined.

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PubMed1997

Morphogenetic potential of the chick leg interdigital mesoderm when diverted from the cell death program.

There is evidence that the interdigital mesoderm may be in an undifferentiated state. For example, under experimental manipulation in vivo it may be diverted from cell death to digit formation. In the present work we wanted to analyze the maximum morphogenetic potential of the interdigital cells. To do this we made recombinant limbs of three types, the first using dissociated-reaggregated leg interdigital mesoderm, the second using the same tissue but without dissociation and the third adding a piece of polarizing region to the dissociated interdigit. In all three the massive cell death of the interdigit failed to occur. The first type of recombinant formed a small nodule of cartilage while the other two formed a well-developed digit. Our data indicate that the maximum morphogenetic potential of the interdigital tissue appears constrained to form digits and that dissociation of the tissue decreased this ability; polarizing region restores the ability of dissociated cell recombinants to form a digit. We also analyzed in these recombinants the expression of a battery of genes implicated in interdigital cell death or in digital morphogenesis. The pattern of expression of each gene analyzed was identical in the three types of recombinant limbs. The expression of Msx1 and Msx2 genes was maintained under the ridge indicating a good interaction between the interdigital cells, both dissociated and undissociated, and the apical ridge. The expression of Hoxd-12, Hoxd-13 and Hoxa-13 genes was maintained in the recombinants, indicating that these cells carry information about their autopodial origin, and this correlates well with their distal restricted morphogenetic potential. Finally, the patterns of expression of the Bmp-2, Bmp-4 and Bmp-7 genes indicated that they are independently regulated in the recombinants and that Bmp-4 and Bmp-7 have wider expression domains than the areas of cell death that were only detected under the regressing apical ridge during day 3 of the experiment.

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PubMed1997

Role of BMP-2 and OP-1 (BMP-7) in programmed cell death and skeletogenesis during chick limb development.

Bone Morphogenetic Protein 2 (BMP-2) and Osteogenic Protein 1 (OP-1, also termed BMP-7) are members of the transforming growth factor beta superfamily. In the present study, we have analyzed the effects of administering them locally at different stages and locations of the chick limb bud using heparin beads as carriers. Our results show that these BMPs are potent apoptotic signals for the undifferentiated limb mesoderm but not for the ectoderm or the differentiating chondrogenic cells. In addition, they promote intense radial growth of the differentiating cartilages and disturb the formation of joints accompanied by alterations in the pattern of Indian hedgehog and ck-erg expression. Interestingly, the effects of these two BMPs on joint formation were found to be different. While the predominant effect of BMP-2 is alteration in joint shape, OP-1 is a potent inhibitory factor for joint formation. In situ hybridizations to check whether this finding was indicative of specific roles for these BMPs in the formation of joints revealed a distinct and complementary pattern of expression of these genes during the formation of the skeleton of the digits. While Op-1 exhibited an intense expression in the perichondrium of the developing cartilages with characteristic interruptions in the zones of joint formation, Bmp-2 expression was a positive marker for the articular interspaces. These data suggest that, in addition to the proposed role for BMP-2 and OP-1 in the establishment of the anteroposterior axis of the limb, they may also play direct roles in limb morphogenesis: (i) in regulating the amount and spatial distribution of the undifferentiated prechondrogenic mesenchyme and (ii) in controlling the location of the joints and the diaphyses of the cartilaginous primordia of the long bones once the chondrogenic aggregates are established.

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PubMed1997

Slug, a zinc finger gene previously implicated in the early patterning of the mesoderm and the neural crest, is also involved in chick limb development.

The great advances made over the last few years in the identification of signalling molecules that pattern the limb bud along the three axes make the limb an excellent model system with which to study developmental mechanisms in vertebrates. The understanding of the signalling networks and their mutual interactions during limb development requires the characterisation of the corresponding downstream genes. In this study we report the expression pattern of Slug, a zinc-finger-containing gene of the snail family, during the development of the limb, and its regulation by distinct axial signalling systems. Slug expression is highly dynamic, and at different stages of limb development can be correlated with the zone of polarizing activity, the progress zone and the interdigital areas. We show that the maintenance of its expression is dependent on signals from the apical ectodermal ridge and independent of Sonic Hedgehog. We also report that, in the interdigit, apoptotic cells lie outside of the domains of Slug expression. The correlation of Slug expression with areas of undifferentiated mesenchyme at stages of tissue differentiation is consistent with its role in early development, in maintaining the mesenchymal phenotype and repressing differentiation processes. We suggest that Slug is involved in the epithelial-mesenchymal interactions that lead to the maintenance of the progress zone.

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PubMed1996

Surgical removal of limb bud Sonic hedgehog results in posterior skeletal defects.

Using Sonic Hedgehog (Shh) as a marker for polarizing region cells we have repeated the experiments of MacCabe et al. (1973) and Fallon and Crosby (1975) in an attempt to reexamine the question of a continuous role for the polarizing region during limb development. We report that the earlier experiments probably left Shh-expressing cells after surgery. Our results show that Shh-expressing cells do not regenerate and complete removal of the polarizing region results in truncations along the anteroposterior (A-P) axis; further, A-P patterning cannot be restored when a bead soaked in FGF is implanted in the limb bud mesenchyme to maintain outgrowth after extirpation of the polarizing region. However, in order to reproducibly remove all Shh-positive cells, it is possible that cells with posterior limb skeletal fate also must be removed. Therefore, microsurgical approaches do not permit an unequivocal answer to the question raised in this and the earlier papers and it remains a reasonable possibility that at least up to stage 20-21 the polarizing region plays a continuous role in patterning of the limb bud during its development.

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PubMed1996

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.

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PubMed1996

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.

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PubMed1996

The limb field mesoderm determines initial limb bud anteroposterior asymmetry and budding independent of sonic hedgehog or apical ectodermal gene expressions.

We have analyzed the pattern of expression of several genes implicated in limb initiation and outgrowth using limbless chicken embryos. We demonstrate that the expressions of the apical ridge associated genes, Fgf-8, Fgf-4, Bmp-2 and Bmp-4, are undetectable in limbless limb bud ectoderm; however, FGF2 protein is present in the limb bud ectoderm. Shh expression is undetectable in limbless limb bud mesoderm. Nevertheless, limbless limb bud mesoderm shows polarization manifested by the asymmetric expression of Hoxd-11, -12 and -13, Wnt-5a and Bmp-4 genes. The posterior limbless limb bud mesoderm, although not actually expressing Shh, is competent to express it if supplied with exogenous FGF or transplanted to a normal apical ridge environment, providing further evidence of mesodermal asymmetry. Exogenous FGF applied to limbless limb buds permits further growth and determination of recognizable skeletal elements, without the development of an apical ridge. However, the cells competent to express Shh do so at reduced levels; nevertheless, Bmp-2 is then rapidly expressed in the posterior limbless mesoderm. limbless limb buds appear as bi-dorsal structures, as the entire limb bud ectoderm expresses Wnt-7a, a marker for dorsal limb bud ectoderm; the ectoderm fails to express En-1, a marker of ventral ectoderm. As expected, C-Lmx1, which is downstream of Wnt-7a, is expressed in the entire limbless limb bud mesoderm. We conclude that anteroposterior polarity is established in the initial limb bud prior to Shh expression, apical ridge gene expression or dorsal-ventral asymmetry. We propose that the initial pattern of gene expressions in the emergent limb bud is established by axial influences on the limb field. These permit the bud to emerge with asymmetric gene expression before Shh and the apical ridge appear. We report that expression of Fgf-8 by the limb ectoderm is not required for the initiation of the limb bud. The gene expressions in the pre-ridge limb bud mesoderm, as in the limb bud itself, are unstable without stimulation from the apical ridge and the polarizing region (Shh) after budding is initiated. We propose that the defect in limbless limb buds is the lack of a dorsal-ventral interface in the limb bud ectoderm where the apical ridge induction signal would be received and an apical ridge formed. These observations provide evidence for the hypothesis that the dorsal-ventral ectoderm interface is a precondition for apical ridge formation.

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PubMed1996

Role of TGF beta s and BMPs as signals controlling the position of the digits and the areas of interdigital cell death in the developing chick limb autopod.

The establishment of the digital rays and the interdigital spaces in the developing limb autopod is accompanied by the occurrence of corresponding domains of expression of TGF beta s and BMPs. This study analyzes whether these coincident events are functionally correlated. The experiments consisted of local administration of TGF beta-1, TGF beta-2 or BMP-4 by means of heparin or Affi-gel blue beads to the chick limb autopod in the stages preceding the onset of interdigital cell death. When beads bearing either TGF beta-1 or -2 were implanted in the interdigits, the mesodermal cells were diverted from the death program forming ectopic cartilages or extra digits in a dose- and stage-dependent fashion. This change in the interdigital phenotype was preceded by a precocious ectopic expression of ck-erg gene around the bead accompanied by down-regulation of bmp-4, msx-1 and msx-2 gene expression. When BMP-beads were implanted in the interdigital spaces, programmed cell death and the freeing of the digits were both accelerated. Implantation of beads bearing BMP-4 at the tip of the growing digits was followed by digit bifurcation, accompanied by the formation of an ectopic area of cell death resembling an extra interdigit, both morphologically and molecularly. The death-inducing effect of the BMP beads and the chondrogenic-inducing effect of the TGF beta beads were antagonized by the implantation of an additional bead preabsorbed with FGF-2, which constitutes a signal characteristic of the progress zone. It is concluded that the spatial distribution of digital rays and interdigital spaces might be controlled by a patterned distribution of TGF beta s and BMPs in the mesoderm subjacent to the progress zone.

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PubMed1995

Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage.

BACKGROUND: Sonic hedgehog (Shh), a vertebrate homolog of the Drosophila segment polarity gene hedgehog (hh), has been implicated in patterning of the developing chick limb. Such a role is suggested by the restricted expression of Shh along the posterior limb bud margin, and by the observation that heterologous cells expressing Shh have limb-polarizing activity resembling that of cells from the polarizing region of the posterior limb bud margin. It has not been demonstrated, however, that the Sonic hedgehog protein (SHH) alone is sufficient for limb patterning. SHH has been shown to undergo autoproteolytic cleavage in vitro, yielding two smaller products. It is of interest, therefore, to determine whether processing of SHH occurs in the developing limb and how such processing influences the function of SHH. RESULTS: We demonstrate that SHH is proteolytically processed in developing chick limbs. Grafts of cells expressing SHH protein variants that correspond to individual cleavage products demonstrate that the ability to induce patterned gene expression and to impose morphological pattern upon the limb bud is limited to the amino-terminal product (SHH-N) of SHH proteolytic cleavage. We also demonstrate that bacterially synthesized and purified SHH-N, released from implanted beads, is sufficient for limb-patterning activity. Finally, we show that the endogenous amino-terminal cleavage product is tightly localized to the posterior margin of the limb bud. CONCLUSIONS: Our data show that, of the two cleavage products resulting from SHH autoproteolysis, SHH-N expressed in grafted heterologous cells or supplied in purified form is sufficient to impose pattern upon the developing limb. Moreover, the restricted localization of the endogenous amino-terminal SHH cleavage product to the posterior border of the chick limb bud makes it unlikely that its patterning activity results from it being distributed in a broad gradient across the antero-posterior axis. More consistent with the observed localization is a model in which the amino-terminal SHH cleavage product exerts its patterning effects by local induction in or near the polarizing region, initiating a cascade of gene expression that ultimately extends across the developing limb.

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PubMed1995

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.

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PubMed1995

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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PubMed1995

Lack of correlation between c-myc expression and programmed or experimentally-induced cell death during chick limb development.

The protooncogene c-myc positively regulates cell death in most in vitro cell model systems under conditions of restricted proliferation, and it has been proposed that cell death is a physiological function of c-myc. To investigate possible changes in c-myc expression concomitant with programmed cell death, we have analyzed c-myc mRNA expression during chick limb development. Expression of c-myc was high in the premuscular masses at earlier stages and in the chondrogenic condensations at later stages of limb development, as demonstrated by in situ mRNA hybridization in sections and whole-mount. However, we did not detect c-myc expression in specific limb areas where massive apoptosis was occurring. Furthermore, when the apical ridge was removed from stage 20 wing buds, there was no increase in the expression of c-myc in the subridge mesoderm, despite the extensive cell death induced by this surgical manipulation. Therefore, our results show no correlation between elevated c-myc expression and either programmed or experimentally-induced apoptosis in the developing limb. These results argue against a role of c-myc in cell death occurring in vivo.

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PubMed1994

FGF-2: apical ectodermal ridge growth signal for chick limb development.

The apical ectodermal ridge permits growth and elongation of amniote limb buds; removal causes rapid changes in mesodermal gene expression, patterned cell death, and truncation of the limb. Ectopic fibroblast growth factor (FGF)-2 supplied to the chick apical bud mesoderm after ridge removal will sustain normal gene expression and cell viability, and allow relatively normal limb development. A bioassay for FGFs demonstrated that FGF-2 was the only detectable FGF in chick limb bud extracts. By distribution and bioactivity, FGF-2 is the prime candidate for the chick limb bud apical ridge growth signal.

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