Metabolic and genomic adaptations to winter fattening in a primate species, the grey mouse lemur (Microcebus murinus).

AIM: To understand the mechanisms underlying the development of metabolic changes leading to obesity remains a major world health issue. Among such mechanisms, seasonality is quite underestimated although it corresponds to the manifestation of extreme metabolic flexibility in response to a changing environment. Nevertheless, the changes induced by such flexibility are far to be understood, especially at the level of insulin signaling, genomic stability or inflammation. METHODS: Here, we investigated the metabolic regulations displayed by a seasonal primate species, the grey mouse lemur (Microcebus murinus) that exhibits pronounced changes in body mass during the 6-month winter season: a fattening period followed by a spontaneous fat loss, without ever reaching pathological stages. RESULTS: Such body weight modulations result from a combination of behavioral (food intake) and physiological (endocrine changes, switch between carb and lipid oxidation) adjustments that spontaneously operate during winter. Conversely to classical models of obesity, insulin sensitivity is paradoxically preserved during the obesogenic phase. Fat loss is associated with increased metabolic activity, especially in brown adipose tissue, and induced increased oxidative stress associated with telomere length dynamic. Furthermore, liver gene expression analysis revealed regulations in metabolic homeostasis (beta-oxidation, insulin signaling, cholesterol and lipid metabolism) but not for genes involved in inflammatory process (for example, Ifng, Tnf, Nfkb1). CONCLUSION: Altogether, these results show that mouse lemurs undergo deep physiological and genomic seasonal changes, without ever reaching a pathological stage. Further investigation is needed to decipher the underlying mechanisms, which may well be highly relevant for human therapeutic strategies.

Hnf1b renal expression directed by a distal enhancer responsive to Pax8.

Xenopus provides a simple and efficient model system to study nephrogenesis and explore the mechanisms causing renal developmental defects in human. Hnf1b (hepatocyte nuclear factor 1 homeobox b), a gene whose mutations are the most commonly identified genetic cause of developmental kidney disease, is required for the acquisition of a proximo-intermediate nephron segment in Xenopus as well as in mouse. Genetic networks involved in Hnf1b expression during kidney development remain poorly understood. We decided to explore the transcriptional regulation of Hnf1b in the developing Xenopus pronephros and mammalian renal cells. Using phylogenetic footprinting, we identified an evolutionary conserved sequence (CNS1) located several kilobases (kb) upstream the Hnf1b transcription start and harboring epigenomic marks characteristics of a distal enhancer in embryonic and adult renal cells in mammals. By means of functional expression assays in Xenopus and mammalian renal cell lines we showed that CNS1 displays enhancer activity in renal tissue. Using CRISPR/cas9 editing in Xenopus tropicalis, we demonstrated the in vivo functional relevance of CNS1 in driving hnf1b expression in the pronephros. We further showed the importance of Pax8-CNS1 interaction for CNS1 enhancer activity allowing us to conclude that Hnf1b is a direct target of Pax8. Our work identified for the first time a Hnf1b renal specific enhancer and may open important perspectives into the diagnosis for congenital kidney anomalies in human, as well as modeling HNF1B-related diseases.

Constrained G4 structures unveil topology specificity of known and new G4 binding proteins.

G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. An essential feature of G4 is their intrinsic polymorphic nature, which is characterized by the equilibrium between several conformations (also called topologies) and the presence of different types of loops with variable lengths. In cells, G4 functions rely on protein or enzymatic factors that recognize and promote or resolve these structures. In order to characterize new G4-dependent mechanisms, extensive researches aimed at identifying new G4 binding proteins. Using G-rich single-stranded oligonucleotides that adopt non-controlled G4 conformations, a large number of G4-binding proteins have been identified in vitro, but their specificity towards G4 topology remained unknown. Constrained G4 structures are biomolecular objects based on the use of a rigid cyclic peptide scaffold as a template for directing the intramolecular assembly of the anchored oligonucleotides into a single and stabilized G4 topology. Here, using various constrained RNA or DNA G4 as baits in human cell extracts, we establish the topology preference of several well-known G4-interacting factors. Moreover, we identify new G4-interacting proteins such as the NELF complex involved in the RNA-Pol II pausing mechanism, and we show that it impacts the clastogenic effect of the G4-ligand pyridostatin.

Binding properties of mono- and dimeric pyridine dicarboxamide ligands to human telomeric higher-order G-quadruplex structures.

Here, we report on the in vitro binding properties of the known pyridine dicarboxamide G-quadruplex ligand 360A and a new dimeric analogue (360A)2A to human telomeric DNA higher-order G-quadruplex (G4) structures. This study points to original binding features never reported for G4 ligands, and reveals a greater efficiency for the dimeric ligand to displace RPA (a ssDNA binding protein involved in telomere replication) from telomeric DNA.

Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes.

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we describe ethidium derivatives that stabilize G-quadruplexes. These molecules were shown to increase the melting temperature of an intramolecular quadruplex structure, as shown by fluorescence and absorbance measurements, and to facilitate the formation of intermolecular quadruplex structures. In addition, these molecules may be used to reveal the formation of multi-stranded DNA structures by standard fluorescence imaging, and therefore become fluorescent probes of quadruplex structures. This recognition was associated with telomerase inhibition in vitro: these derivatives showed a potent anti-telomerase activity, with IC(50) values of 18-100 nM in a standard TRAP assay.

Interaction of human DNA topoisomerase I with G-quartet structures.

Because of their role in the control of the topological state of DNA, topoisomerases are ubiquitous and vital enzymes, which participate in nearly all events related to DNA metabolism including replication and transcription. We show here that human topoisomerase I (Topo I) plays an unexpected role of 'molecular matchmaker' for G-quartet formation. G-quadruplexes are multi-stranded structures held together by square planes of four guanines ('G-quartets') interacting by forming Hoogsteen hydrogen bonds. Topo I is able to promote the formation of four-stranded intermolecular DNA structures when added to single-stranded DNA containing a stretch of at least five guanines. We provide evidence that these complexes are parallel G-quartet structures, mediated by tetrads of hydrogen-bonded guanine. In addition, Topo I binds specifically to pre-formed parallel and anti-parallel G4-DNA.

Differential effects of amsacrine and epipodophyllotoxins on topoisomerase II cleavage in the human c-myc protooncogene.

Amsacrine and demethylepipodophyllotoxins (etoposide and teniposide) are potent topoisomerase II inhibitors which have optimum activity in different cancers. To investigate whether these differences are due to different activity on cellular oncogenes, drug-induced topoisomerase II cleavage sites were mapped and sequenced in the human c-myc protooncogene. In the presence of purified murine L1210 topoisomerase II, amsacrine induces prominent cleavage in the P2 promoter (site 2499/2502). Footprinting experiments indicate that topoisomerase II binds to the entire promoter region (approximately 20 base pairs on the sides of the P2 site). In the case of teniposide or etoposide, cleavage is more diffuse and markedly less at the P2 site. Mapping of cleavage sites in human small cell lung carcinoma cells (NCI N417) also shows that cleavage in the P2 promoter region is induced preferentially by amsacrine but not by demethylepipodophyllotoxins. Thus, selective gene damage among topoisomerase II inhibitors may contribute to differential anticancer activity.

Molecular and cellular interactions between intoplicine, DNA, and topoisomerase II studied by surface-enhanced Raman scattering spectroscopy.

The surface-enhanced Raman scattering spectra of the new antitumoral agent, intoplicine (RP 60475, NSC 645008), and those of its complexes with DNA and topoisomerase II in vitro and in K562 cancer cells were obtained. Intoplicine was found to unwind DNA and to inhibit purified calf thymus topoisomerase II via a stabilization of the ternary cleavable complex. The intensity of the surface-enhanced Raman scattering spectrum of intoplicine was not modified by the addition of plasmid pBR322 or calf thymus DNA. In the complex of this antitumor agent with topoisomerase II, the signal of intoplicine was completely abolished, indicating that at least some portion of intoplicine binds to an internal part of the enzyme. During the formation of the ternary complex, intoplicine was released from the interior of the protein and formed hydrogen bonds via its hydroxyl and/or amino groups. Similar modifications of the intoplicine spectra were found by microsurface-enhanced Raman scattering spectroscopy of the compound in the nucleus of treated K562 cells. In contrast, intoplicine was found to be in a free form in the cytoplasm.

Specific inhibition of serine- and arginine-rich splicing factors phosphorylation, spliceosome assembly, and splicing by the antitumor drug NB-506.

Specific phosphorylation of serine- and arginine-rich pre-mRNA splicing factors (SR proteins) is one of the key determinants regulating splicing events. Several kinases involved in SR protein phosphorylation have been identified and characterized, among which human DNA topoisomerase I is known to have DNA-relaxing activity. In this study, we have investigated the mechanism of splicing inhibition by a glycosylated indolocarbazole derivative (NB-506), a potent inhibitor of both kinase and relaxing activities of topoisomerase I. NB-506 completely inhibits the capacity of topoisomerase I to phosphorylate, in vitro, the human splicing factor 2/alternative splicing factor (SF2/ASF). This inhibition is specific, because NB-506 does not demonstrate activity against other kinases known to phosphorylate SF2/ASF such as SR protein kinase 1 and cdc2 kinase. Importantly, HeLa nuclear extracts competent in splicing but not splicing-deficient cytoplasmic S100 extracts treated with the drug fail to phosphorylate SF2/ASF and to support splicing of pre-mRNA substrates containing SF2/ASF-target sequences. Native gel analysis of splicing complexes revealed that the drug affects the formation of the spliceosome, a dynamic ribonucleoprotein structure where splicing takes place. In the presence of the drug, neither pre-spliceosome nor spliceosome is formed, demonstrating that splicing inhibition occurs at early steps of spliceosome assembly. Splicing inhibition can be relieved by adding phosphorylated SF2/ASF, showing that extracts treated with NB-506 lack a phosphorylating activity required for splicing. Moreover, NB-506 has a cytotoxic effect on murine P388 leukemia cells but not on P388CPT5 camptothecin-resistant cells that carry two point mutations in conserved regions of topoisomerase I gene (Gly361Val and Asp709Tyr). After drug treatment, P388 cells accumulated hypophosphorylated forms of SR proteins and polyadenylated RNA in the nucleus. In contrast, neither SR protein phosphorylation nor polyadenylated mRNA distribution was affected in P388 CPT5-treated cells. Consistently, NB506 treatment altered the mRNA levels and/or splicing pattern of several tested genes (Bcl-X, CD 44, SC35, and Sty) in P388 cells but not in P388 CPT5 cells. The study shows for the first time that indolocarbazole drugs targeting topoisomerase I can affect gene expression by modulating pre-mRNA splicing through inhibition of SR proteins phosphorylation.

Poisoning of topoisomerase I by an antitumor indolocarbazole drug: stabilization of topoisomerase I-DNA covalent complexes and specific inhibition of the protein kinase activity.

We have investigated the mechanism of topoisomerase I inhibition by an indolocarbazole derivative, R-3. The compound is cytotoxic to P388 leukemia cells, but not to P388CPT5 camptothecin-resistant cells having a deficient topoisomerase I. R-3 can behave both as a specific topoisomerase I inhibitor trapping the cleavable complexes and as a nonspecific inhibitor of a DNA-processing enzyme acting via DNA binding. In addition, the drug is a potent inhibitor of the kinase activity of topoisomerase I. Unlike camptothecin, R-3 completely inhibits the phosphorylation of SF2/ASF, a member of the SR protein family, in the absence of DNA. The inhibitory effect is also observed using mutant enzyme Y723F that lacks DNA cleavage/religation activity but does not affect phosphotransferase activity, indicating, therefore, that R-3 acts independently at both DNA cleavage and protein kinase sites. R-3 is the only compound known thus far that interferes specifically with the kinase activity of topoisomerase I and not with other kinases, such as protein kinase C and the cdc2 kinase. The study reinforces the view that topoisomerase I is a dual enzyme with a DNA cleavage site juxtaposed to a functionally independent kinase site and shows for the first time that indolocarbazole drugs can inhibit both the DNA cleavage/religation and kinase activities of the enzyme.

Intoplicine (RP 60475) and its derivatives, a new class of antitumor agents inhibiting both topoisomerase I and II activities.

Intoplicine (RP 60475, NSC 645008) is an antitumor derivative in the 7H-benzo[e]pyrido[4,3-b]indole series which is now being tested in clinical trials. Intoplicine strongly binds DNA (KA = 2 x 10(5) M-1) and thereby increases the length of linear DNA. These properties are consistent with DNA unwinding by intoplicine. Intoplicine was found to be a dual topoisomerase I and II inhibitor, with DNA sites of enzyme inhibition being different for these two enzymes. In this study, 22 analogues of intoplicine were evaluated for their effects on topoisomerase I- and II-mediated DNA cleavage reactions by using enzymes purified from calf thymus. Site-specific DNA cleavage mediated by topoisomerase I was observed with 7H-benzo[e]pyrido[4,3-b]indole derivatives but not with 11H-benzo[g]-pyrido[4,3-b]indole derivatives. Site-specific DNA cleavage mediated by topoisomerase II occurred with derivatives having hydroxyl groups at the 3-position on the 7H-benzo[e]pyrido[4,3-b]indole ring or at the 4-position on the 11H-benzo[g]pyrido[4,3-b]indole ring. Study of the relationships between the in vivo antitumor activity on P388 leukemia and the topoisomerase I- and/or II-mediated DNA cleavage activity revealed that the most highly active antitumor compounds possessed both topoisomerase I-and II-inhibitory properties. Compounds selectively inhibiting either topoisomerase I or II were less active. These results suggest that dual topoisomerase I and II inhibition is critical for the antitumor activity of this new series of antitumor compounds.

Identification and properties of a major plasma metabolite of irinotecan (CPT-11) isolated from the plasma of patients.

Irinotecan [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11)] is a promising water-soluble analogue of camptothecin [S. Sawada et al., Chem. & Pharm. Bull. (Tokyo), 39: 1446-1454, 1991]. We have reported previously the presence of an important polar metabolite, in addition to 7-ethyl-10-hydroxycamptothecin (SN-38) beta-glucuronide, in samples of plasma taken from patients undergoing treatment with CPT-11 (L.P. Rivory and J. Robert, Cancer Chemother. Pharmacol. 36: 176-179, 1995; L. P. Rivory and J. Robert, J. Cromatogr., 661: 133-141, 1994). Plasma samples (0.5 ml) containing comparatively large amounts of this metabolite were extracted by solid-phase columns and subjected to high-performance liquid chromatography and mass spectrometry in parallel to fluorometric detection. The metabolite yielded [M + 1] ions with a m/z of 619, representing the addition of 32 atomic mass units to CPT-11. Purified fractions were subjected to proton nuclear magnetic resonance, and the structure determined, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycampothecin (APC), was further validated following synthesis. Like CPT-11, APC was found to be only a weak inhibitor of the cell growth of KB cells in culture (IC50, 2.1 versus 5.5 micrograms/ml for CPT-11 and 0.01 microgram/ml for SN-38, the active metabolite of CPT-11) and was a poor inducer of topoisomerase I DNA-cleavable complexes (100-fold less potent than SN-38). In contrast to CPT-11, APC was not hydrolyzed to SN-38 by human liver microsomes or purified human liver carboxylesterase. Furthermore, APC did not inhibit the hydrolysis of CPT-11 in these preparations. Interestingly, APC was only a weak inhibitor of acetylcholinesterase in comparison to CPT-11 and neostigmine. It appears likely, therefore, that APC does not contribute directly to the activity and toxicity profile of CPT-11 in vivo.

[Telomeres and telomerase, new targets for anticancer chemotherapy]. / Télomères et télomérase, de nouvelles cibles pour la chimiothérapie anticancéreuse.

Telomeres are composed of single-strand DNA rich in guanine which can adopt particular structures such as T-loop or G-quadruples, a four-strand DAN structure formed by guanine repeats. Telomeric single-strand DNA is the substrate of telomerase, an enzyme necessary for telomeric replication which is suppressed in most cancer cells and which participates in tumor genesis. The formation of a telomeric G-quadruplex blocks telomerase activity and offers an original strategy for new anti-cancer agents. Using an original approach combining rational screening and synthesis, several series of compounds have been identified which specifically bind to the telomeric quadruplex. These derivatives, called "G-quadruplex DNA ligands", are able to block telomeric replication in cancer cells and provoke replicative senescence and/or apoptosis after a few cell cycles. Our team is working on characterizing the cellular and molecular mechanisms of action of these ligands. Using mutant cell models resistant to these ligands or expressing a protein cuff covering the telomere in tumor lines, we have demonstrated that the telomere is the principal intracellular target of action of these compounds and the implicit existence of the G-quadruplex structure. In collaboration with academic and industrial partners, optimization of these ligands to develop pharmacologically active products should enable in vivo validation of a new therapeutic concept.

Human ARF protein interacts with topoisomerase I and stimulates its activity.

The ARF gene (p19(ARF) in mouse and p14(ARF) in man) has become a central actor of the cell cycle regulation process as it participates to the ARF-MDM2-p53 pathway and the Rb-E2F-1 pathway. By use of immunoprecipitation and Western blotting (IP/WB), we now show that ARF physically associates with topoisomerase I (Topo I). ARF-Topo I immune complexes were detected in SF9 insect cells infected with recombinant baculoviruses encoding the two genes as well as in 293 cells that express endogenously these proteins. Preparations of a GST-ARF recombinant protein stimulated the DNA relaxation activity of Topo I but, in contrast, had no effect on the decatenation activity of Topo II. The Topo I stimulation was also detected in cell extracts of SF9 cells expressing both proteins. A confocal microscopy study indicated that part of ARF and Topo I colocalized in the granular component structure of the nucleolus. As a whole, our data indicate that Topo I is a new partner of ARF and suggest that ARF is involved in cell reactions that require Topo I.

Structure-activity relation in camptothecin antitumor drugs: why a detailed molecular characterisation of their lactone and carboxylate forms by Raman and SERS spectroscopies?

Lactone and carboxylate forms of potent antitumor agents, camptothecins (CPTs) have been studied by Raman, Fourier-transform Raman (FT-Raman) and surface-enhanced Raman scattering (SERS) spectroscopy. Similarity of the Raman spectra of CPTs with corresponding FT-Raman spectra in the near-infrared allowed the latter to be compared with their SERS counterparts in order to analyse the interaction of the drugs with silver colloids. Different types of silver colloids (reduced with sodium borohydride or sodium citrate, with or without activation by anions) have been evaluated. Citrate-reduced colloid, activated with chloride anions (CAS) has been found to be the best compromise for SERS studies of both forms of CPTs. We suggest that in general CPTs are adsorbed on CAS via the nitrogen in ring B and are more inclined to a flat orientation than to a perpendicular one. However, probable interactions of substitution groups and/or of the COO- groups of hydrolysed CPTs with the CAS surface introduce some particularities in the adsorption patterns. As a result, SERS spectra are highly sensitive to hydrolysis and substitutions at distant rings of CPT and uniquely characteristic of each of the CPT derivatives. The pronounced hydrolysis-induced changes, similar in the Raman and SERS spectra of CPTs, involve similar vibrations in the spectra of different CPTs. Vibrational assignments, proposed for the main Raman and SERS bands of CPT and its derivatives (21-lactam-S-CPT, 10,11-(methylenedioxy)-CPT, CPT-11, SN-38 and topotecan) indicate that most of the bands which decreased upon lactone hydrolysis are those preferentially related to stretching modes of the quinoline rings A and B, and the bands which increased are those of the ring D stretching modes. Our data make the spectroscopic approach very promising for the further investigations of the molecular mechanisms of biological activity of CPTs.

Kinetics of lactone hydrolysis in antitumor drugs of camptothecin series as studied by fluorescence spectroscopy.

Potent antitumor activity exhibited by 20-S-camptothecin (CPT) and numerous derivatives is known to be lost upon opening of the alpha-hydroxy-lactone ring of these drugs, hydrolyzable at neutral and basic pH. To quantify in 'real time' the lactone hydrolysis reaction in CPTs under physiological conditions, we have applied a non-perturbing approach by fluorescence spectroscopy. CPT and a set of its derivatives (21-lactam-S-CPT, 10,11-(methylenedioxy)-CPT, CPT-11, SN-38, topotecan, tricyclic ketone-CPT) with antitumor activity varying from negligible to 10 times that of CPT have been studied. Prior to the kinetic measurements, the effects of substitutions, pH, polarity of molecular environment, lactone ring opening (lactone-carboxylate transition) have been investigated in terms of the UV-visible absorption and fluorescence emission spectra of CPTs. Then the determined parameters of the fluorescence emission spectra corresponding to the respective lactone and carboxylate forms have been used to estimate the residual lactone percentage as a function of time. The reproducibility of the obtained data demonstrates that the spectroscopic approach provides a satisfactory precision for this kind of measurements. For CPT at pH 7.3, the lactone half-life was 29.4 +/- 1.7 min and the lactone percentage at equilibrium was 20.9 +/- 0.3%. Within a series of derivatives with substitutions at quinoline rings, the lactone half-life varied from 29 to 32 min and the equilibrium lactone content varied from 15% to 23%. For each compound, even slight increase of pH from 7.1 to 7.3 or from 7.3 to 7.6 logically leads to a remarkable decrease of both lactone half-life and equilibrium lactone percentage.

Role of fibroblast growth factor during early midbrain development in Xenopus.

Genes encoding fibroblast growth factors (FGFs) are expressed in early Xenopus neurulae in the prospective midbrain-hindbrain boundary (MHB) region of the neural plate. These expression domains overlap those of XWnt-1 and XEn-2, raising the question of the role of FGF signalling in the regulation of these genes, and more generally about the function of FGF during Xenopus midbrain development. We report that explants from the prospective MHB grafted into the anterior neural plate in midneurula stage embryos induce XWnt-1 expression and, at a lower frequency, XEn-2 expression in the vicinity of the graft. Such a process is likely to involve FGF signalling. Implantation of FGF4- or FGF8-soaked beads in the prospective forebrain at neurula and tailbud stages causes the up-regulation of XWnt-1 and XEn-2 in the dorsal and lateral region of the anterior midbrain. This effect is not relayed by endogenous FGF genes since exogenous FGFs inhibit the expression of endogenous XFGF3 or XFGF8. However, consequences of grafting MHB or implanting FGF4 or FGF8 beads on tadpole brain development are different. MHB grafts induce ectopic mesencephalic structures, strongly suggesting that a region homologous to the isthmic organizer of amniotes is specified as early as the midneurula stage. In contrast, exogenous FGFs do not cause the formation of ectopic mesencephalic structures but an overgrowth of mesencephalon and diencephalon. We propose that FGF signals from the prospective MHB play a crucial role in the spatial regulation of XWnt-1 and XEn-2 expression in the posterior midbrain, but that the full organizing activity of the MHB involves other factors in combination with FGF.

Cloning and developmental expression of STAT5 in Xenopus laevis.

Transcription factors of the signal transducer and activator of transcription (STAT) family are required for cellular responses to multiple signalling molecules. After ligand binding-induced activation of cognate receptors, STAT proteins are phosphorylated, hetero- or homodimerize, and enter the nucleus. STAT dimers bind to specific DNA elements and alter the transcriptional activity of the signal-responsive genes. We report the cloning and developmental pattern of expression of XSTAT5, a Xenopus laevis member of the STAT family, closely related to the mammalian STAT5A and STAT5B. XSTAT5 is expressed maternally and zygotically. With the onset of neurulation, XSTAT5 RNA are clearly localized in the anterior neural plate and subsequently in the neural structures of the developing eye, the pineal gland and the cement gland anlage. At late tailbud stages, a faint expression is detected in a ventral location that might correspond to the ventral blood islands.

What mechanisms drive cell migration and cell interactions in Pleurodeles?

Embryogenesis implies a strict control of cell interaction and cell migration. The spatial and temporal regulation of morphogenetic movements occurring during gastrulation is directly dependent on the early cell interactions that take place in the blastula. The newt Pleurodeles waltl is a favorable model for the study of these early morphogenetic events. The combination of orthotopic grafting and fluorescent lineage tracers has led to precise early gastrula mesoderm fate maps. It is now clear that there are no sharp boundaries between germ layers at the onset of gastrulation but rather diffuse transition zones. The coordination of cell movements during gastrulation is closely related to the establishment of dorsoventral polarity. Ventralization by U.V. irradiation or dorsalization by lithium treatment modifies the capacity for autonomous migration on the fibronectin coated substratum of marginal zone cells accordingly. It is now firmly established that mesodermal cells need to adhere to a fibrillar extracellular matrix (ECM) to undergo migration during gastrulation. Extracellular fibrils contain laminin and fibronectin (FN). Interaction of cells with ECM involves receptors of the beta 1 integrin family. A Pleurodeles homolog of the alpha v integrin subunit has been recently identified. Protein alpha v expression is restricted to the surface of mesodermal cells during gastrulation. Integrin-mediated interactions of cells with FN are essential for ECM assembly and mesodermal cell migration. Intracellular injection of antibodies to the cytoplasmic domain of beta 1 into early cleavage embryos causes inhibition of FN fibril formation. Intrablastocoelic injections of several probes including antibodies to FN or integrin alpha 5 beta 1, competitive peptides to the major cell binding site of FN or the antiadhesive protein tenascin all block mesodermal cell migration. This results in a complete arrest of gastrulation indicating that mesodermal cell migration is a major driving force in urodele gastrulation. It is now possible to approach the role of fibroblast growth factor (FGF) during cell interactions taking place in urodele embryos. Four different FGF receptors (FGFR) have been cloned in Pleurodeles. Each of them has a unique mRNA expression pattern. FGFR-1, FGFR-3, and the variant of FGFR-2 containing the IIIb exon are maternally expressed and might be involved in mesodermal induction. During gastrulation, FGFR-3 and FGFR-4 have a restricted pattern of expression, whereas FGFR-1 mRNA is nearly uniformly distributed. Splicing variants FGFR-2IIIb and FGFR-2IIIc have exclusive expression patterns during neurulation. IIIb is expressed in epidermis and IIIc in neural tissue, suggesting a function in the differentiation of ectodermal derivatives.

Fibronectin-rich fibrillar extracellular matrix controls cell migration during amphibian gastrulation.

We have reviewed the evidence supporting the notion that the fibrillar extracellular matrix on the basal surface of the blastocoel roof in amphibian embryos directs and guides mesodermal cell migration during gastrulation. Based on extensive experimental evidence in several different systems, we conclude the following: (i) the fibrillar extracellular matrix contains fibronectin (FN) and laminin. (ii) The fibrils are oriented in such a way as to promote directional migration of mesodermal cells during migration. (iii) We have used several different probes to disrupt the interaction between migrating mesodermal cells and the fibrillar extracellular matrix. These probes include: (a) nucleocytoplasmic and interspecific hybridization. Such embryos have defects in FN synthesis and gastrulation. (b) Fab' fragments of anti-FN and anti-integrin VLA-5 IgGs prohibit mesodermal cell adhesion both in vitro and in vivo and gastrulation is arrested. (c) Peptides containing the RGDS sequence specifically inhibit interactions between migrating mesodermal cells and the FN-fibrillar matrix. (d) Tenascin blocks cell adhesion to FN in vitro and gastrulation in vivo. (e) Antibodies against the cytoplasmic domain of beta 1 integrin, when injected into blastomeres, prevent FN-fibrillogenesis in progeny of injected blastomeres and delay mesodermal cell migration selectively in the progeny of injected blastomeres but not in the uninjected blastomere progeny.