Molecular cloning and embryonic expression of the Xenopus Arnt gene.

In this paper, we report the cloning of a Xenopus bHLH/PAS factor homologous to the mammalian aryl hydrocarbon receptor nuclear translocator (Arnt) or Drosophila Tango gene. Sequence data analysis indicates that protein domains organization in xArnt is strongly conserved and that xArnt is highly related to the mammalian Arnt1 isoform. As revealed by reverse transcriptase polymerase chain reaction and whole-mount in situ hybridization, xArnt gene is expressed during early and late development. At early stages, xArnt transcripts are restricted to the ectoderm and extends to the marginal zone at gastrula stage. In tail bud embryo, xArnt is strongly expressed in branchial arches, optical and optical vesicles, and pronephros and pronephritic duct.

Characterization and developmental expression of xSim, a Xenopus bHLH/PAS gene related to the Drosophila neurogenic master gene single-minded.

We have isolated a novel gene from Xenopus, denominated xSim, which encodes a protein of 760 amino acids containing a basic helix-loop-helix (bHLH) motif contiguous to a PAS domain characteristic of an emerging family of transcriptional regulators so called bHLH/PAS. xSim shares a strong amino acid sequence identity with the Drosophila Single-minded (dSim) and with the murine Sim1 and Sim2 proteins. Phylogenetic analysis reveals that xSim gene is an ortholog gene of the mSim2 gene. Spatio-temporal analysis shows a maternal and a zygotic expression of xSim throughout early Xenopus development. In situ hybridization assays reveal that the transcripts are enriched in the animal hemisphere until blastula stage and extend to the marginal zone at early gastrula stage. As development proceeds, xSim is mainly restricted to the central nervous system.

HSP70 is involved in the control of chromosomal transcription in the amphibian oocyte.

The amphibian oocyte represents an excellent model for the study of transcription regulation. Indeed, any modification of transcriptional activity is directly reflected in lampbrush chromosome structure by concomitant morphological changes. Previous studies have led to the hypothesis of a putative role for heat-shock proteins HSP70 and/or HSC70 in transcriptional processes in the oocyte. In order to dissect out the relative role of HSP70 or HSC70 in these processes, we used an oligo-antisense strategy to specifically inhibit the function of the targeted protein. Effects of hsc70 and hsp70 antisense oligodeoxynucleotides were analyzed in terms of both mRNA quantity and protein synthesis. Their effects on oocyte transcription were analyzed at the level of structural organization of lampbrush chromosomes and nucleolar transcriptional activity. Our results show that specific inactivation of hsc70 mRNA by hsc70 antisense oligos led to a reversible inhibition of lampbrush chromosome transcription. However, such reversible inhibition of transcription is considered non-sequence specific since it is also induced by any oligo. In contrast, specific inactivation of hsp70 mRNA by hsp70 antisense oligos, which is correlated with a drop of HSP70 neosynthesis, results in an irreversible inhibition of lampbrush chromosome transcription. Furthermore, our results show that the inactivation of hsp70 or hsc70 mRNAs does not affect nucleolar transcription. Such data suggest a role for HSP70 in the control of chromatin modifications related to RNA polymerase II transcriptional activity.

Cell-cycle-dependent nuclear translocation of HSP70 in amphibian embryonic cells.

Using immunocytochemical methods, we analyzed the localization of the HSP70 protein constitutively expressed during embryogenesis in the amphibian Pleurodeles waltl. Our results provide evidence for nuclear transfer of the protein during gastrulation, and particularly for predominant nuclear labeling in gastrula internalized cells. Using two inhibitors of DNA replication -hydroxyurea (HUA) and aphidicolin- or/and an inhibitor of transcription -actinomycin D- applied to embryos, we demonstrated that nuclear transfer of HSP70 is related to the transcriptional activity of the cells during the early S phase of the cell cycle.

Evidence for specific RNA/protein interactions in the differential segment of the W sex chromosome in the amphibian Pleurodeles waltl.

Pleurodeles exhibits a ZZ/ZW system of GSD (genotype sex determination). However, the Z and W sex chromosomes appear to be morphologically identical. A short RNA sequence is described that was specifically bound to lampbrush loops in the differential segment of the sexual bivalent IV. The distribution of these labeled loops in experimentally produced ZZ and WW females enabled us to demonstrate that such labeled loops were perfectly correlated with the W chromosome. Therefore, this RNA sequence constitutes an excellent marker for the W differential segment. Furthermore, analysis of the labeled loops under various experimental conditions suggested that their labeling is caused by specific interactions between this RNA sequence and lampbrush loop-associated proteins (RNA/protein interactions). North-western assays revealed that nuclear polypeptide(s) of 65 kDa could be responsible for such binding.

Molecular cloning of a cDNA encoding the amphibian Pleurodeles waltl 70-kDa heat-shock cognate protein.

We isolated and characterized a cDNA coding for heat-shock protein 70 of the amphibian Pleurodeles waltl. This 2212-bp sequence exhibited one open reading frame of 645 amino acids. The predicted amino acid sequence exhibited the three conserved elements of the HSC/HSP70 protein family. Comparison of nucleotide and amino acid sequences between this gene and other hsc/hsp-like genes revealed a high identity with the cognate form HSC70. By in vitro translation, this gene encoded a 70-kDa protein which was different than the inducible Pleurodeles waltl HSP70 protein. This translated protein was recognized by Pleurodeles waltl N1 anti-HSC/HSP70 antibody. Heat-inducibility tests showed that this gene was constitutively expressed during oogenesis and embryogenesis, and its expression was not increased after a heat-shock. These results led us to conclude that we recovered a Pleurodeles waltl cognate hsc70 gene.

Conditions for a heat shock response during oogenesis and embryogenesis of the amphibian Pleurodeles waltl.

The optimal conditions capable of inducing an increase in HSP70 neosynthesis during development of the urodele amphibian Pleurodeles waltl were determined in this study. These conditions depend on temperature, heat shock duration and recovery duration. In oocytes, a heat shock response was repeatedly obtained at 37 degrees C for 15 min followed by 1 h recovery. These results provided evidence for heat shock response at every stage considered. An increase in HSP70 synthesis was noted throughout oogenesis, but it did not lead to an increase in the amount of soluble HSP70, except for stage VI oocytes. Such results suggest that from stage II to stage IV oocytes, an equilibrium occurs between the HSP70 used and the HSP70 neosynthesized. In contrast, in stage VI oocytes, heat shock led to overproduction of HSP70. During early development, the heat shock response was repeatedly obtained only from the gastrula stage with a 37 degrees C shock and a 15 min duration of treatment. Surprisingly, during cleavage stage, the soluble HSP70 total amount increased after heat shock at a time when no HSP70 neosynthesis occurred.

Molecular characterization of a heat shock cognate cDNA of zebrafish, hsc70, and developmental expression of the corresponding transcripts.

To elucidate the potential role of the hsp70 gene family in developmental processes in vertebrates, we chose to study the expression of one of these genes in zebrafish. A zebrafish gastrula cDNA library was screened with a Pleurodeles waltl hsp70 cDNA probe. A 2.3-kb cDNA was thus isolated and sequenced. The predicted amino acid sequence contained an open reading frame encoding for a 649-amino acid polypeptide. Sequence analysis showed strong homology with hsp70-related gene sequences in other species; in particular, the strongest homology was found with the cognate members of this family. Tests of heat inducibility revealed that transcripts were expressed at normal temperature, but the level of transcript expression increased after heat shock. Moreover, experiments of the neosynthesis of total proteins in heat shock conditions and corresponding immunoblotting assays showed that 24-h-stage embryos are able to respond to heat shock. The quantity of 70 kDa proteins, recognized by a specific antibody of the HSP/C70 protein family, is expressed in control condition and increased significantly after heat shock. Furthermore, Northern blot analysis of transcript expression showed that the corresponding mRNAs were detected throughout embryonic development in the absence of any heat shock. Our clone, named hsc70, thus corresponded to a cognate member of the hsp70 gene family, expressed under normal conditions during development, but also heat inducible. The spatio-temporal pattern of transcripts during development was determined by in situ hybridization on wholemount embryos at different stages. As a maternal RNA, hsc70 mRNA was uniformly present in the embryo, up to the end of gastrulation. Later, a tissue-specific enrichment of hsc70 transcripts was detected in the central nervous system (CNS) and in a fraction of the somites. These results suggest that the hsc70 gene may be involved in developmental differentiation events.

Tissue-specific expression of an hsc90 gene and nuclear translocation of the HSC90-related protein during amphibian embryogenesis.

Expression and distribution of a constitutive member of the 90 kDa heat-shock protein family, named HSC90, was investigated during amphibian embryonic development. By Northern blot analysis, two hsp90 transcripts (2.5 and 3 kb) which displayed differing developmental regulation were detected during embryogenesis. Expression of the larger transcript (3 kb), which encodes an HSC90-related protein, decreased until the gastrula stage. However, zygotic transcription for this hsc90 gene was found to start from the neurula stage, and the corresponding zygotic hsc90 transcript was specifically located by whole mount in situ hybridization in the anterior neural tube of a late neurula embryo. Later, in a tailbud embryo, hsc90 transcripts were detected in the cephalic region, neural tube, eye vesicles, branchial and mandibular arches and somites. Distribution of the HSC90-related protein was also analysed by immunohistochemistry throughout embryogenesis. As expected, the protein was strongly expressed in the cytoplasm, mainly in the periplasmic area of embryonic tissue cells. Interestingly, HSC90 was also transiently detected in the nuclear area, with this nuclear transfer depending on the chromatin condensation state, up to the blastula stage. During the process of gastrulation, nuclear translocation of HSC90 was also observed at the level of the blastopore dorsal lip, exclusively in cells undergoing invagination.

What role might lampbrush chromosomes play in maternal gene expression?

The biological significance of lampbrush chromosomes from urodelan amphibians is far from being elucidated. Their particularly well developed lateral loops are the site of intense transcriptional activity, which can be visualized in electron microscopy using the Miller spreading procedure. All transcription units functioning in lampbrush loops synthesize RNA at a maximum rate. In situ hybridization has provided evidence for transcription of both unique coding sequences and highly repetitive sequences. The role of lampbrush transcripts in the production of maternal information remains unclear. RNAs transcribed from unique coding sequences are exported to the cytoplasm; there, they contribute either to maintaining the required level of maternal messenger RNA in a basal state during late oogenesis, or to increasing the store of these maternal RNAs throughout oocyte growth, i.e., until stage VI. For repetitive sequences, their intense transcription appears to be non-productive, in that RNAs are not translatable and might be useless products of readthrough transcription. The non-productive transcription of repetitive sequences, the expression of which is directly related to hyperdevelopment of lateral loops, raises the issue of the role of lampbrush chromosome transcription.

Does the chaperone heat shock protein hsp70 play a role in the control of developmental processes?

Expression of an hsp70 gene strictly inducible in somatic cells and constitutively expressed during oogenesis was investigated during embryogenesis of the amphibian Pleurodeles waltl. Results from Northern hybridization experiments and RNase protection assays provided evidence for the presence of inducible hsp70 mRNA under normal conditions at every embryonic stage. Immunoblotting of embryo proteins separated by 2D-electrophoresis provided evidence for the presence of a single polypeptide of about 74 kDa likely to be an HSP70-related protein, from unfertilized egg to tailbud stage. Immunocytological analysis showed that HSP70-related proteins were localized in the cytoplasm of all blastomeres. It also pointed out that nuclear transfer of the protein occurs in certain cells, precisely at the time of their invagination and subsequent internalization during normal Pleurodeles development. Such nuclear transfer involves involuting mesodermal cells in the blastopore region at the time of gastrulation. It also involves neurodermic cells at the time of neural tube closure. Interestingly, in exogastrulas nuclear transfer did not occur in cells which could no longer invaginate. Such behavior of HSP70-related proteins led us to suggest that they are involved in the control of nuclear activity associated with important developmental events such as cellular internalization processes. Such a role may be a direct consequence of HSP70-related protein functional properties as molecular chaperones.

Evidence for a 90 kDa heat-shock protein gene expression in the amphibian oocyte.

In order to study expression of a 90-kDa heat-shock protein during amphibian oogenesis at physiological temperature, we isolated a Pleurodeles waltl hsc90 cDNA by screening an ovarian cDNA library with a chicken hsp90 cDNA probe. The cDNA thus obtained--named Pw90--shows a high homology level with the hsp90 gene in other species. RNase protection analysis led us to conclude that this sequence is part of the cognate gene hsc90 and is constitutively expressed in oocytes. Furthermore, results of quantitative Northern blot analysis, as well as in situ hybridizations on oocyte sections or lampbrush chromosome spreads, provide evidence for expression of hsc90 transcripts at every stage of oogenesis. Moreover, they point to the fact that an accumulation of transcripts occurs very early in oogenesis. Simultaneously, the expression of HSC90-related protein was analyzed on Western blots using a monoclonal antibody (AC88) and a polyclonal antibody (AP90Ct) raised against the Pleurodeles C-terminal part of HSC90. We provide evidence for a net accumulation of HSC90-related protein in oocytes. Immunolocalization shows that a nuclear transfer occurs in the course of oogenesis and leads to a concentration equilibrium between cytoplasm and nucleus in stage VI oocytes.

Transcription of amphibian lampbrush chromosomes is disturbed by microinjection of HSP70 monoclonal antibodies.

To investigate the possible involvement of HSP70 in nuclear transport of proteins associated with the transcription process in amphibian oocyte lampbrush chromosomes, we examined the effect of anti-HSP70 mouse monoclonal antibodies on the transcriptional activity of lampbrush chromosomes. When injected into the oocyte cytoplasm, anti-HSP70 induced disorganization followed by retraction of chromosomal lateral loops. This retraction reflected inhibition of transcription, which was confirmed by our electron microscopic observations. At the same time, while nucleoli were morphologically disorganized, nucleolar transcription persisted. These modifications were completely reversible. In contrast, no modifications were observed when antibodies were directly microinjected into nuclei. Similar observations were registered at the level of lampbrush chromosomes when wheat germ agglutinin was injected into oocyte cytoplasm. All of these results suggest that HSP70 mediates the nuclear transport of proteins involved in the lampbrush chromosome transcriptional process.

Constitutive expression of a somatic heat-inducible hsp70 gene during amphibian oogenesis.

We isolated and characterized a sequence coding for heat-shock protein 70 (HSP70) of the amphibian Pleurodeles waltl. Results from S1 nuclease protection assays led us to conclude that an hsp70 gene, strictly inducible in somatic cells during heat shock, is constitutively active during oogenesis. By quantitative northern and western blot analysis, we showed that both hsp70 mRNA and HSP70-related protein levels increased in oocytes from stage II to stage VI under physiological conditions. Furthermore, by in situ hybridization to the nascent transcripts of lampbrush chromosome loops, we provided evidence for a clear-cut relationship between this increase in hsp70 mRNA and transcriptional activity during the lampbrush stage of oogenesis. These results strongly suggest that hsp70 genes are actively transcribed throughout oogenesis. HSP70-related proteins localized in the cytoplasm of young oocytes are progressively transferred to the nucleus in the course of oogenesis and preferentially accumulated in the nuclei of some stage VI oocytes.

Immunolocalization of HSP 70-related proteins constitutively expressed during Xenopus laevis oogenesis and development.

Using immunocytochemical and biochemical methods, we analyzed the localization of HSP 70-related proteins constitutively expressed during oogenesis and embryogenesis in the amphibian Xenopus laevis. Our results provided evidence for a regional localization in oocytes. In embryos, the regional distribution observed in oocytes was found to be maintained from fertilization up to late blastula. It is noteworthy that, at the beginning of gastrulation, nuclear transfer of such proteins had already occurred by the time of internalization in the involuting marginal zone (IMZ), whereas cells of the vegetal area exhibited only a perinuclear localization of these proteins. These results suggest that HSP 70-related proteins might be involved in the control of the process of cellular internalization.

Electron microscopic in situ hybridization in amphibian lampbrush chromosomes.

Electron microscopy RNA/RNA in situ hybridization was adjusted to Pleurodeles lampbrush chromosomes. The cRNA probe used was synthesized from genomic DNA sequences which were proven to be moderately repetitive. Preembedding hybridization was performed on lampbrush chromosome preparations, and several fixation and hybridization conditions were tested. Paraformaldehyde and glutaraldehyde were used as fixatives at various concentrations and durations. Hybridization was then performed with or without formamide for various times of incubation. The best results were obtained when hybridization was carried out for 4 h at 42 degrees C in the presence of formamide, with lampbrush chromosomes having been previously fixed overnight in a mixture of paraformaldehyde/glutaraldehyde. Due to the excellent preservation of the ultrastructure and specificity of the signal obtained under these conditions, we were able to demonstrate, at the ultrastructural level, that such RNA expression occurs in two types of lampbrush loop ribonucleoprotein matrices showing a different morphology of their transcripts. Furthermore, a different mapping of the same sequence along the loop DNA axes is strongly suggested by a different labeling distribution in these loops.

Microdissection and cloning of DNA from landmark loops of amphibian lampbrush chromosomes.

Microdissection of the "globular" and "granular" landmark loops of Pleurodeles lampbrush chromosomes and subsequent cloning of their DNA yielded several recombinant clones. The 6.6-kb insert of one of them was subcloned and the 600 bp of one subclone was characterized by Southern and slot hybridizations as well as by sequencing. This sequence, designated p130B, was shown to belong to a class of moderately repetitive DNA. RNA expression of this sequence was investigated by in situ hybridization of p130B to the nascent transcripts of lateral loops. Results showed that: (1) the same transcripts were not always found in matrices of landmarks exhibiting the same morphological features; (2) the same transcripts were expressed in loops of different morphological types. Based on these results we suggest that even if there is a morphological similarity of landmark loops, this does not reflect total similarity of their transcripts.

Comparison between in vivo and in vitro heat-induced changes in amphibian lampbrush chromosomes.

At normal breeding temperature (20 degrees C), amphibian lampbrush chromosomes are characterized by the presence of lateral loops which are related to the transcriptional process. Heat treatment induces changes in these loops, but the nature and timing of these modifications depend on hyperthermic stress conditions. Indeed, our data demonstrate that, at the same high temperature (34 degrees C), lampbrush chromosome modifications induced by in vivo and in vitro gradual heat treatments are different from those induced by in vitro heat shock. In vivo and in vitro heat treatments lead to progressive disorganization of landmark loops, whereas in vitro heat shock results in chromosome condensation. The progressive adaptation of lampbrush chromosome structure in response to gradual heat stress is considered and discussed.

Effects of cold shock treatment on lampbrush chromosomes of amphibian oocytes.

When females of the newt Pleurodeles waltl, normally raised in our laboratory at 20 degrees C, were placed in 8 degrees C water for several days, striking modifications occurred in the structure of oocyte lampbrush chromosomes: numerous normal-type lateral loops were partially reduced in size and number, while hyperdeveloped loops of a new morphological type occurred at constant, reproducible loci. However, induction of these cold loops apparently was not accompanied by preferential RNA synthesis at their levels. Cold loops resulted from the decompaction of specific landmarks, the granular loops. Autoradiography revealed a significant drop in lampbrush chromosome transcriptional activity at 8 degrees C. Both structural and transcriptional modifications were fully reversible when oocytes were returned to normal temperature. These modifications are discussed in relation to processes which could determine the morphological appearance of landmark loops.

Chromosomes of amphibian oocytes as a model for gene expression: significance of lampbrush loops.

Amphibian lampbrush chromosome loops exhibit morphological variability in their RNP matrix. The biological significance of such variability remains unknown. In order to approach this problem, the structural organization of each RNP matrix type was analyzed in relation to transcriptional and post-transcriptional processes. First, autoradiographic and transcription inhibition studies in conjunction with macromolecular spread analysis revealed a particular transcription pattern in the most typical loops, i.e. the globular loops. This pattern was characterized by asynchronous variations in RNA synthesis in the different transcription units present in a given loop. Second, morphological and experimental studies provided evidence that the typical morphologies of different RNP matrices were interconvertible and that the differences between the different RNP matrices resulted from various degrees of tightness in packaging of transcription products. In particular, analysis of thermic-shock-induced changes in the structure of lampbrush chromosomes enabled us to visualize the progressive disorganization of dense RNP matrices into globular, granular and normal matrices. Furthermore, these studies suggested that changes in post-transcriptional processes might play a determining role in the specific morphology of the loops. In particular, the kinetics of each of these different processes, related to one another and/or proteins specific to one or another of these processes, might determine the morphological appearance of the loops. The immunological approach revealed that specific nuclear proteins might therefore interfere with each of these processes. Third, the problem of a possible relationship between the specific morphologies of lateral loops and the expression of particular DNA sequences was approached at the molecular level.