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P Ferretti

Publications and source records attributed to P Ferretti.

At least 19 recordsLinked to original sources
PubMed2005

Changes in response to spinal cord injury with development: vascularization, hemorrhage and apoptosis.

Chick embryos are capable of functional spinal cord regeneration following crush injury until embryonic day 13. Developmental changes occurring thereafter result in failure to regenerate. Secondary injury mechanisms can result in apoptotic cell death and make a major contribution to cell loss after trauma. We report here that around embryonic day 13 there is a dramatic increase in blood vessel numbers in the spinal cord, and that the extent of hemorrhage in response to injury increases with developmental age. This is paralleled by increased apoptosis and subsequent cavitation in spinal cords injured at embryonic day 15 as compared with embryonic day 11. Following spinal cord injury at embryonic day 15, apoptotic cell death is extensive and spreads to the same extent as the hemorrhage. When hemorrhage is reduced by treatment with the hemostatic drug desmopressin the extent of apoptosis and cavity formation in spinal cords injured at embryonic day 15 decreases. Furthermore, manipulations of embryonic day 11 spinal cords that increase hemorrhage also increase apoptosis and result in cavitation in contrast to the effective repair typical of this stage. Altogether these results suggest that cavity formation occurring at developmental stages non-permissive for regeneration is largely due to changes in the extent of apoptosis that are related to vascularization and hemorrhage.

Animals↗
PubMed2005

Gene expression during palate fusion in vivo and in vitro.

UNLABELLED: Failure of secondary palate fusion during embryogenesis is a cause of cleft palate. Disappearance of the medial epithelial seam (MES) is required to allow merging of the mesenchyme from both palatal shelves. This involves complex changes of the medial edge epithelial (MEE) cells and surrounding structures that are controlled by several genes whose spatio-temporal expression is tightly regulated. We have carried out morphological analyses and used a semi-quantitative RT-PCR technique to evaluate whether morphological changes and modulation in the expression of putative key genes, such as twist, snail, and E-cadherin, during the fusion process in palate organ culture parallel those observed in vivo, and show that this is indeed the case. We also show, using the organotypic model of palate fusion, that the down-regulation of the transcription factor snail that occurs with the progression of palate development is not dependent on fusion of the palatal shelves. ABBREVIATIONS: dsg1, desmoglein1; EMT, epithelial-mesenchymal transition; MEE, medial edge epithelium; MES, medial epithelial seam; RT-PCR, reverse-transcriptase polymerase chain-reaction.

Animals↗
PubMed2003

Expression of FGF2 in the limb blastema of two Salamandridae correlates with their regenerative capability.

Limb regenerative potential in urodeles seems to vary among different species. We observed that Triturus vulgaris meridionalis regenerate their limbs significantly faster than T. carnifex, where a long gap between the time of amputation and blastema formation occurs, and tried to identify cellular and molecular events that may underlie these differences in regenerative capability. Whereas wound healing is comparable in the two species, formation of an apical epidermal cap (AEC), which is required for blastema outgrowth, is delayed for approximately three weeks in T. carnifex. Furthermore, fewer nerve fibres are present distally early after amputation, consistent with the late onset of blastemal cell proliferation observed in T. carnifex. We investigated whether different expression of putative blastema mitogens, such as FGF1 and FGF2, in these species may underlie differences in the progression of regeneration. We found that whereas FGF1 is detected in the epidermis throughout the regenerative process, FGF2 onset of expression in the wound epidermis of both species coincides with AEC formation and initiation of blastemal cell proliferation, which is delayed in T. carnifex, and declines thereafter. In vitro studies showed that FGF2 activates MCM3, a factor essential for DNA replication licensing activity, and can be produced by blastemal cells themselves, indicating an autocrine action. These results suggest that FGF2 plays a key role in the initiation of blastema growth.

Amputation Stumps↗
PubMed2002

Differential regulation of fibroblast growth factor receptors in the regenerating amphibian spinal cord in vivo.

Unlike mammals, adult urodele amphibians can regenerate their spinal cord and associated ganglia, but the molecular mechanisms controlling regeneration are not fully understood. We have recently shown that expression of FGF2, a member of the fibroblast growth factor family, is induced in the progenitor cells of the regenerating spinal cord and appears to play a role in their proliferation and possibly in their differentiation. In order to investigate which receptor(s) may mediate FGF2 signaling and their role in regeneration, we have studied expression of the four fibroblast growth factor receptors, FGFR1, FGFR2, FGFR3 and FGFR4, and of the spliced variants, sFGFR and KGFR, in the regenerating spinal cord of the adult urodele, Pleurodeles waltl, following tail amputation. We show that all FGFRs are expressed in normal and regenerating spinal cord, with the exception of the spliced variants that are expressed only in non-neural tissues of the tail. FGFR1 and 4 show the more interesting spatio-temporal patterns of expression. They are not detectable in the ependymal cells of normal cords, from which neural progenitors for regeneration are believed to originate, though they are expressed in some mature neurons. During regeneration, significant up-regulation of FGFR1 precedes that of FGFR4 in the ependymal tube from which the new cord will form. FGFR4 is highly expressed in these cells at later stages of regeneration, when neuronal differentiation is becoming apparent, and like FGFR1 is also expressed in some newborn neurons. In addition to the known form of FGFR1, the antibody against this receptor reacts also with a non-phosphorylated protein that appears to be present only during regeneration, and might represent a yet undescribed variant of the receptor. Altogether this study shows that fibroblast growth factor signaling is finely modulated during tail and spinal cord regeneration, and points to FGFR1 and FGFR4 as key players in this process, suggesting that FGFR1 is primarily associated with proliferation of progenitor cells and FGFR4 with early stages of neuronal differentiation.

Animals↗
PubMed2001

FGF2 promotes skeletogenic differentiation of cranial neural crest cells.

The cranial neural crest gives rise to most of the skeletal tissues of the skull. Matrix-mediated tissue interactions have been implicated in the skeletogenic differentiation of crest cells, but little is known of the role that growth factors might play in this process. The discovery that mutations in fibroblast growth factor receptors (FGFRs) cause the major craniosynostosis syndromes implicates FGF-mediated signalling in the skeletogenic differentiation of the cranial neural crest. We now show that, in vitro, mesencephalic neural crest cells respond to exogenous FGF2 in a dose-dependent manner, with 0.1 and 1 ng/ml causing enhanced proliferation, and 10 ng/ml inducing cartilage differentiation. In longer-term cultures, both endochondral and membrane bone are formed. FGFR1, FGFR2 and FGFR3 are all detectable by immunohistochemistry in the mesencephalic region, with particularly intense expression at the apices of the neural folds from which the neural crest arises. FGFRs are also expressed by subpopulations of neural crest cells in culture. Collectively, these findings suggest that FGFs are involved in the skeletogenic differentiation of the cranial neural crest.

Animals↗
PubMed2000

FGF-2 Up-regulation and proliferation of neural progenitors in the regenerating amphibian spinal cord in vivo.

Regeneration of the spinal cord occurs spontaneously in adult urodele amphibians. The key cells in this regenerative process appear to be the ependymal cells that following injury migrate and proliferate to form the ependymal tube from which the spinal cord regenerates. Very little is known about the signal(s) that initiates and maintains the proliferative response of these cells. Fibroblast growth factor 2 (FGF-2) has been shown to play a role in maintaining neural progenitor cell cycling in vitro and may be important for neuronal survival and axonal growth after injury. We have investigated its role in regeneration of the spinal cord in vivo following tail amputation in the adult salamander, Pleurodeles waltl. We show that only the low-molecular-weight form of FGF-2 is found in Pleurodeles and that in the normal cord it is expressed in a subset of neurons, but is hardly detectable in ependymal cells. Tail amputation results in induction of FGF-2 in the ependymal cells of the regenerating structure, and later in regeneration FGF-2 is up-regulated in some newborn neurons. FGF-2 pattern of expression in the ependymal tube parallels that of proliferation. Furthermore, exogenous FGF-2 significantly increases ependymal cell proliferation in vivo. Overall our results strongly support the view that one important role of FGF-2 during spinal cord regeneration in Pleurodeles is to induce proliferation of neural progenitor cells.

Amputation, Surgical↗
PubMed1999

RA regulation of keratin expression and myogenesis suggests different ways of regenerating muscle in adult amphibian limbs.

Formation of a regeneration blastema following limb amputation is believed to occur through a process of dedifferentiation. It has been suggested, however, that the cells contributed to the blastema by the stump muscle are satellite-like cells, rather than cells originated by dedifferentiation. We have previously shown that simple epithelial keratins 8 and 18 are expressed in the mesenchymal progenitor cells of the regenerating amphibian limb and in cultured cells with myogenic potential, and that their expression appears to be causally related to changes in proliferation and differentiation. We show here that retinoic acid (RA) affects the expression of these keratins differently in myogenic cells originated from normal limb and limb blastema. Furthermore, we find that the effects of RA on proliferation, myogenic differentiation and adhesion of these cells also differ. In fact, whereas RA does not affect keratin expression, proliferation or myogenic differentiation in blastemal cells, it does decrease keratin levels and thymidine incorporation and increase myogenesis in cells from normal limb. Conversely, RA increases cell adhesion only in blastemal cells. Significantly, these effects of RA on cultured cells are consistent with those observed in vivo. Overall the results presented here suggest that in the urodele limb there are two distinct cell populations with myogenic potential, one originating from dedifferentiation and one equivalent to the satellite cells of the mammalian muscle, which are likely to be primarily involved in blastema formation and muscle repair, respectively.

Amputation Stumps↗
PubMed1998

Stability and plasticity of neural crest patterning and branchial arch Hox code after extensive cephalic crest rotation.

The extent to which the spatial organisation of craniofacial development is due to intrinsic properties of the neural crest is at present unclear. There is some experimental evidence supporting the concept of a prepattern established within crest while contiguous with the neural plate. In experiments in which the neural tube and premigratory crest are relocated within the branchial region, crest cells retain patterns of gene expression appropriate for their position of origin after migration into the branchial arches, resulting in skeletal abnormalities. But in apparent conflict with these findings, when crest is rerouted by late deletion of adjacent crest, infilling crest alters its pattern of gene expression to match its new location, and a normal facial skeleton results. In order to reconcile these findings thus identify processes of relevance to the course of normal development, we have performed a series of neural tube and crest rotations producing a more extensive reorganisation of cephalic crest than has been previously described. Lineage analysis using DiI labelling of crest derived from the rotated hindbrain reveals that crest does not migrate into the branchial arch it would have colonised in normal development, rather it simply populates the nearest available branchial arches. We also find that crest adjacent to the grafted region contributes to a greater number of branchial arches than it would in normal development, resulting in branchial arches containing mixed cell populations not occurring in normal development. We find that after exchange of first and third arch crest by rotation of r1-7, crest alters its expression of hoxa-2 and hoxa-3 to match its new location within the embryo resulting in the reestablishment of the normal branchial arch Hox code. A facial skeleton in which all the normal components are present, with some additional ectopic first arch structures, is formed in this situation. In contrast, when second and third arch crest are exchanged by rotation of r3 to 7, ectopic Hox gene expression is stable, resulting in the persistence of an abnormal branchial arch Hox code and extensive defects in the hyoid skeleton. We suggest that the intrinsic properties of crest have an effect on the spatial organisation of structures derived from the branchial arches, but that exposure to increasingly novel environments within the branchial region or "community effects" within mixed populations of cells can result in alterations to crest Hox code and morphogenetic fate. In both classes of operation we find that there is a tight link between the resulting branchial arch Hox code and a particular skeletal morphology.

Animals↗
PubMed1998

Hedgehog family member is expressed throughout regenerating and developing limbs.

Members of the hedgehog family have been shown to play a key role in many developmental processes, including limb patterning and chondrogenesis. We have therefore investigated whether members of this family are also expressed during regeneration of the adult urodele limb and are regulated by retinoic acid (RA), since this derivative induces proximodistal duplications in regenerating limbs, and has been shown to regulate sonic hedgehog (shh) in the developing limbs of birds and mammals. We report here that a newt homologue of Xenopus banded hedgehog, called N-bhh, is uniformly expressed by mesenchymal blastemal cells from the initial stages of regeneration and is up-regulated by RA. In addition, we show that N-bhh is uniformly expressed in the early limb bud of the newt embryo. Since bhh has not been detected in developing limbs of higher vertebrates, its expression in developing and regenerating newt limbs may be related to the regenerative capability of urodeles.

Amino Acid Sequence↗
PubMed1998

Gene expression during amphibian limb regeneration.

Limb regeneration in adult urodeles is an important phenomenon that poses fundamental questions both in biology and in medicine. In this review, we focus on recent advances in the characterization of the regeneration blastema at cellular and molecular levels and on the current understanding of the molecular basis of limb regeneration and its relationship to development. In particular, we discuss (i) the spatiotemporal distribution of genes and gene products in the mesenchyme and wound epidermis of the regenerating limb, (ii) how growth is controlled in the regeneration blastema, and (iii) molecules that are likely to be involved in patterning the regenerating limb such as homeobox genes and retinoids.

Animals↗
PubMed1998

Segmentation, crest prespecification and the control of facial form.

The early development of the vertebrate head is dependent on the formation of two series of segmented structures, the rhombomeres of the hindbrain and the branchial arch series. The initial formation of these two systems is closely linked, as the principal source of branchial arch mesenchyme is the neural crest, which derives from the lateral edge of the neural plate at the time of rhombomere formation. The subsequent development of the two systems maintains a close level of integration, as specific spatial relationships between skeletal, muscle and neural elements arising from the same axial level are established. Given the level of conservation of these anatomical relationships in vertebrates, it is likely that they are a reflection of a key mechanism in early facial and pharyngeal development. One model, in part based on these findings, proposed that the neural crest acquires an axial-level specific combination of gene expression while part of the neural plate. This prepattern is then maintained throughout the crest's subsequent development. In the model, this combination of gene expression would then specify the form of the facial and pharyngeal structures that the crest would give rise to. In this review we evaluate recent evidence on whether early facial development involves a crest prespecification of this type, and conclude that it is not the case.

Animals↗
PubMed1997

A role for midline closure in the reestablishment of dorsoventral pattern following dorsal hindbrain ablation.

The cellular and molecular study of dorsal neural tube ablation reported here demonstrates a critical role for midline closure in hindbrain repatterning. This was revealed by detailed analysis of the transcriptional response of two genes, Pax-3 and slug, during repair of the neural tube following ablation. The reexpression of Pax-3 appears to rely on a single surface ectoderm/neuroepithelial contact, while this is insufficient for reexpression of slug. In fact, slug up-regulation only occurred upon midline closure and, strikingly, corresponded to down-regulation of Pax-3. We examined whether a candidate dorsalizing molecule, Bmp-4, was responsible for this reciprocal regulation of Pax-3 and slug at midline closure. However, Bmp-4 was not reexpressed following ablation, indicating not only that it is not responsible for the observed repatterning but that it lies in regulatory pathways distinct from Pax-3 and slug. We additionally examined the expression of Pax-6, which, together with assessment of the pattern of cranial ganglia, roof plate morphology, and positioning of branchiomotor exit points, demonstrates that neural crest regeneration is accompanied by reestablishment of a normal dorsoventral pattern within the neural tube. Thus, both local and longer range patterning appears to be restored following ablation, which is reliant dorsally on midline closure of the neural tube.

Animals↗
PubMed1997

Expression of regeneration-associated cytoskeletal proteins reveals differences and similarities between regenerating organs.

The unique events which allow regeneration of an entire organ to occur are formation of a specialized wound epidermis and accumulation of progenitor cells (blastemal cells) at the amputated surface to form a blastema. In order to identify some of the molecular events underlying the early stages of the regenerative process which are either common to different systems or specific to one of them, we have investigated whether molecules which are induced in limb blastemas are also expressed in skin repair and during regeneration of other complex body structures (lower jaws, upper jaws, and tails). In addition, we have addressed the issue of the identity of progenitor cells during jaw development and regeneration by analyzing the expression of limb blastemal markers in the developing head and face. We have focused on cytoskeletal components, and particularly on the epidermal keratin NvKII, the simple epithelial keratins 8 and 18 and 22/18, because they are among the few molecules which have been shown to be associated with regeneration in the limb and may play significant roles in various developmental processes. Some important findings emerge from this study: 1) Expression of the epidermal keratin NvKII, unlike that of its mammalian homologue K6, is not simply induced in response to wounding, but is associated with regeneration of specific organs. In fact, NvKII is expressed in regenerating limbs and tails, but not in upper or in lower jaw regenerates, demonstrating the existence of molecular differences in the composition of the wound epidermis in these systems. This, together with the fact that NvKII mRNA is regulated by retinoic acid, which differentially affects patterning of limbs and jaws, argues for distinct inductive abilities of the wound epidermis in different organs. 2) In contrast to the differential expression of the epidermal keratin NvKII, the regeneration-associated cytoskeletal molecules identified in limb blastemal cells are expressed in a similar fashion in jaw and tail blastemas. Therefore, it appears that similar cellular events lead to the establishment of an actively proliferating population of progenitor cells from the stump of different organs. Finally, the mesenchyme of the facial rudiments, unlike that of developing limb buds, expresses simple epithelial keratins. Thus, it appears that mesenchymal progenitor cells of developing and regenerating jaws are alike in regard to their intermediate filament content, and this may be related to nerve-dependent growth control of progenitor cells in different developing and regenerating systems.

Animals↗
PubMed1997

Keratin 8 and 18 expression in mesenchymal progenitor cells of regenerating limbs is associated with cell proliferation and differentiation.

Keratins are considered markers of epithelial differentiation. In lower vertebrates, however, immunoreactivity for keratin 8 and 18 has been reported in nonepithelial cells, particularly in mesenchymal progenitor cells of regenerating complex body structures. To confirm that such reactivity does indeed reflect keratin expression and to investigate their possible role in regeneration, we have isolated clones coding for the newt homologues of keratin 8 and 18 (NvK8 and NvK18, respectively) and studied their distribution and changes in their expression following experimental manipulations. Analysis of NvK8 and NvK18 transcripts confirms that K8 and K18 are expressed in the blastemal cells of regenerating newt limbs and that their expression is first observed 3-5 days after amputation, when the blastemal cells start to proliferate under the influence of the nerve, whose presence is essential for regeneration to proceed. In contrast, no induction of these keratins is observed following amputation of a larval limb at a stage when organogenesis is proceeding in a nerve-independent manner. To establish whether there is a causal relationship between keratin expression and cell proliferation in the adult limb blastema, we have investigated whether their expression is nerve-dependent and whether suppression of their expression in cultured blastemal cells affects cell division and differentiation. Analysis of keratins in denervated limbs demonstrates that the nerve is not necessary to induce their expression. However, treatment of cultured blastemal cells with K8 and K18 anti-sense oligonucleotides significantly decreases DNA synthesis and induces changes in cell morphology, suggesting that expression of these keratins during regeneration may be necessary for the maintenance of the undifferentiated and proliferative state of blastemal cells.

Amino Acid Sequence↗
PubMed1997

Correlation between RA-induced apoptosis and patterning defects in regenerating fins and limbs.

We have compared the ability of RA to induce apoptosis in regenerating fins and limbs in order to establish whether there may be a possible causal relationship between RA-induced cell death and the different patterning abnormalities observed in these two systems following RA treatment. In regenerating fins RA affects the anteroposterior axis and induces narrowing of the fin and fusion of rays. In regenerating limbs however, it mainly affects the proximodistal axis, where dose-dependent duplications of segments are observed. We report here that RA increases cell death by apoptosis both in fin and limb regenerates independently from the route of administration, but in distinct cell populations. In regenerating fins, RA-induced apoptosis is observed only in the wound epidermis, whereas in regenerating limbs significant apoptosis is observed mainly in the blastema mesenchyme. Altogether, these findings support the view that RA-induced apoptosis is causally related to the patterning defects observed in fins and limbs.

Animals↗
PubMed1997

Expression of the transcription factor slug correlates with growth of the limb bud and is regulated by FGF-4 and retinoic acid.

The slug gene encodes a zinc finger transcription factor expressed by neural crest cells (Nieto et al., Science 264: 835-839, 1994) and by certain non-crest derived mesenchymal cell populations, such as lateral mesoderm and sclerotome (Mayor et al., Development 121: 767-777, 1995; Buxton et al., Dev. Biol. 183: 150-165, 1997). We report here that slug is also expressed in developing chick limbs. The slug expression domain in the limb bud expands from posterior to anterior and marks cells that are predominantly destined to become chondrocytes but have not yet differentiated. Its expression is maintained in connective tissue, but is never observed in the premuscle masses. We show that removal of the apical ectodermal ridge results in loss of slug expression which can be arrested by the addition of an FGF-4 bead. Retinoic acid bead implants lead to down-regulation of slug expression, again accompanied by abolition of limb outgrowth. Dual bead implants demonstrate antagonism between these two factors, suggesting that a localized antagonistic effect between endogenous RA and FGF-4 on slug expression underlies the molecular mechanism controlling the transition between undifferentiated and differentiated state during normal limb development. The fact that slug expression pattern correlates with areas of growth in the limb, and is maintained by FGF-4 and down-regulated by retinoic acid, indicates that slug-expressing cells play a crucial role in growth and patterning of the chick limb. We propose that slug expression provides the best correlation to date between a molecular marker and the physical concept of the progress zone, defined as "a labile region where new positional values are successively engendered in the course of growth" (Summerbell et al., Nature 244: 492-496, 1973).

Animals↗
PubMed1996

Re-examining jaw regeneration in urodeles: what have we learnt?

Urodele amphibians can regenerate not only their limbs and tails, but also their upper and lower jaws rather faithfully. However, relatively few studies of jaw regeneration in amphibians have been carried out, especially in recent years. It is therefore important to reexamine thoroughly this regenerating system, since the advent of sophisticated morphological techniques and the development of molecular approaches offer the promise of renewed and rapid progress in our understanding of complex developmental problems such as this. This paper briefly reviews some of the early research on jaw regeneration, some of the fundamental questions which have been asked and have yet to be answered, and the work we have carried out in order to understand the molecular mechanisms underlying jaw regeneration in the newt, Notophthalmus viridescens. In addition, some aspects of jaw regeneration will be discussed in relation to regeneration of the adult limb.

Animals↗