A role for SOX9 in post-transcriptional processes: insights from the amphibian oocyte.

Sox9 is a member of the gene family of SOX transcription factors, which is highly conserved among vertebrates. It is involved in different developmental processes including gonadogenesis. In all amniote species examined thus far, Sox9 is expressed in the Sertoli cells of the male gonad, suggesting an evolutionarily conserved role in testis development. However, in the anamniotes, fishes and amphibians, it is also expressed in the oocyte but the significance of such an expression remains to be elucidated. Here, we have investigated the nuclear localization of the SOX9 protein in the oocyte of three amphibian species, the urodelan Pleurodeles waltl, and two anurans, Xenopus laevis and Xenopus tropicalis. We demonstrate that SOX9 is associated with ribonucleoprotein (RNP) transcripts of lampbrush chromosomes in an RNA-dependent manner. This association can be visualized by Super-resolution Structured Illumination Microscopy (SIM). Our results suggest that SOX9, known to bind DNA, also carries an additional function in the posttranscriptional processes. We also discuss the significance of the acquisition or loss of Sox9 expression in the oocyte during evolution at the transition between anamniotes and amniotes.

Effect of cadmium on gonadogenesis and metamorphosis in Pleurodeles waltl (urodele amphibian).

In the amphibian Pleurodeles waltl, steroid hormones play a key role in sex differentiation. Since cadmium has been reported to block receptors of sex steroid hormones, we analyzed the effects of this heavy metal on Pleurodeles larvae gonadogenesis. At stage 42, larvae die in the presence of 10.9 microM Cd in the rearing tap water, with TL(50) of 46.3 h, but the concentration of 5.5 microM is tolerated for more than 60 days. When used at 5.5 microM cadmium accumulation measured by atomic absorption spectrophotometry (AAS) in total homogenates of larvae at stage 54 (after 77 days of exposure to the heavy metal) reached 58.1 microg/g of dry weight. At stage 54, we did not detect inhibitory effects on gonadogenesis in larvae reared in the presence of 5.5 microM Cd since stage 42. When the exposure to 5.5 microM Cd was lengthened after stage 54, metamorphosis was delayed and could not be completed. When larvae were exposed to 10.9 microM Cd from stage 54, metamorphosis did not occur and gonad development was stopped. Our study demonstrates a lack of a direct effect of cadmium on sex determination-differentiation but a strong inhibitory effect on metamorphosis, which impairs further gonadal development.

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.

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.

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.

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.

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.

Structure of landmark loop ribonucleoprotein matrices in Pleurodeles waltlii lampbrush chromosomes visualized by scanning electron microscopy.

Landmark loop ribonucleoprotein (RNP) matrices of Pleurodeles waltlii lampbrush chromosomes were systematically examined by scanning electron microscopy (SEM). The results, which corroborated similar studies by electron microscopy (EM), showed that RNP transcripts in normal loops, and RNP matrices in granular, globular and dense loops, are composed of one basic structure: an RNP particle with a diameter of 30 nm. SEM observations also clarified the spatial arrangement of this particle in the RNP matrices of all the loop types examined. The specific morphology of normal, granular, globular and dense loop RNP matrices depended on the degree of compaction of the transcription products; this compaction resulted both from the packaging of RNP transcripts and the progressive coiling of the loop axis.

Size uniformity of ribonucleoprotein matrix particles in loops of Pleurodeles waltlii lampbrush chromosomes visualized by electron microscopy.

The different kinds of loops of lampbrush chromosomes were identified in phase contrast, then analysed by electron microscopy on thin sections. Examination at high magnification showed that the basic structure of the ribonucleoprotein (RNP) matrix of all kinds of loops is a 30 nm RNP particle. Furthermore, this study suggests that the morphological differences between the loops are due to the extent of aggregation of these particles.

Evidence for a particular mode of transcription in globular loops of lampbrush chromosomes of the newt Pleurodeles waltlii.

In amphibian lampbrush chromosomes, many loops have a specific morphology; this is the case for globular loops in the newt Pleurodeles. We have previously shown that the specific morphology of these loops is linked to an extreme compactness of the transcription products which make up their matrix. We investigated RNA synthesis in this type of loop by carrying out autoradiographic and transcription inhibition studies. We also analysed the organization of transcriptional complexes in these loops in the electron microscope using spread preparations. These studies revealed the presence of several transcription units in the same loop and asynchronous variations in RNA synthesis in these transcription units. We propose and discuss several hypotheses in order to explain this asynchronous RNA synthesis. We also discuss these results in the context of loop morphology and transcription mode.