Lineage tracing of sclerotome cells in amphibian reveals that multipotent somitic cells originate from lateral somitic frontier.

The two somite compartments, dorso-lateral dermomyotome and medio-ventral sclerotome are major vertebrate novelties, but little is known about their evolutionary origin. We determined that sclerotome cells in Xenopus come from lateral somitic frontier (LSF) by lineage tracing, ablation experiments and histological analysis. We identified Twist1 as marker of migrating sclerotome progenitors in two amphibians, Xenopus and axolotl. From these results, three conclusions can be drawn. First, LSF is made up of multipotent somitic cells (MSCs) since LSF gives rise to sclerotome but also to dermomytome as already shown in Xenopus. Second, the basic scheme of somite compartmentalization is conserved from cephalochordates to anamniotes since in both cases, lateral cells envelop dorsally and ventrally the ancestral myotome, suggesting that lateral MSCs should already exist in cephalochordates. Third, the transition from anamniote to amniote vertebrates is characterized by extension of the MSCs domain to the entire somite at the expense of ancestral myotome since amniote somite is a naive tissue that subdivides into sclerotome and dermomyotome. Like neural crest pluripotent cells, MSCs are at the origin of major vertebrate novelties, namely hypaxial region of the somite, dermomyotome and sclerotome compartments. Hence, change in MSCs properties and location is involved in somite evolution.

The phosphoinositide 3-kinase pathway and glycogen synthase kinase-3 positively regulate the activity of metal-responsive transcription factor-1 in response to zinc ions.

Metal-responsive transcription factor-1 (MTF-1) is a metal-regulatory transcription factor essential for induction of the genes encoding metallothioneins (MTs) in response to transition metal ions. Activation of MTF-1 is dependent on the interaction of zinc with the zinc fingers of the protein. In addition, phosphorylation is essential for MTF-1 transactivation. We previously showed that inhibition of phosphoinositide 3-kinase (PI3K) abrogated Mt expression and metal-induced MTF-1 activation in human hepatocellular carcinoma (HCC) HepG2 and mouse L cells, thus showing that the PI3K signaling pathway positively regulates MTF-1 activity and Mt gene expression. However, it has also been reported that inhibition of PI3K has no significant effects on Mt expression in immortalized epithelial cells and increases Mt expression in HCC cells. To further characterize the role of the PI3K pathway on the activity of MTF-1, transfection experiments were performed in HEK293 and HepG2 cells in presence of glycogen synthase kinase-3 (GSK-3), mTOR-C1, and mTOR-C2 inhibitors, as well as of siRNAs targeting Phosphatase and TENsin homolog (PTEN). We showed that inhibition of the mTOR-C2 complex inhibits the activity of MTF-1 in HepG2 and HEK293 cells, while inhibition of the mTOR-C1 complex or of PTEN stimulates MTF-1 activity in HEK293 cells. These results confirm that the PI3K pathway positively regulates MTF-1 activity. Finally, we showed that GSK-3 is required for MTF-1 activation in response to zinc ions.

Multiple sequences and factors are involved in stability/degradation of Awnt-1, Awnt-5A and Awnt-5B mRNAs during axolotl development.

Following fertilization in amphibian, early cleavage stages are maternally controlled at a post-transcriptional level before initiation of zygotic transcriptions at the mid blastula transition (MBT). We document the expression levels of the axolotl Awnt-1, Awnt-5A and Awnt-5B genes as well as the adenylation states of their corresponding mRNAs from the end of oogenesis until the tailbud stages. Awnt-1/-5A RNAs are stable until MBT then degraded before gastrulation. Awnt-5B RNAs are degraded at fertilization and zygotically expressed after MBT with high level expression from gastrulation. Estimation of the poly(A) tail lengths reveals no direct link between deadenylation and degradation periods for each Awnt transcript. To investigate the molecular mechanisms involved in Awnt-1/-5A/-5B RNAs stability, synthetic full-length or 3' untranslated region (UTR) Awnt RNAs progressively deleted from their 3' end were microinjected in axolotl oocytes, unfertilized and fertilized eggs. We identified degrading and stabilizing sequences in the 3'UTR whose activities depend on the cellular context and are also modulated by the 5'UTR and coding sequence within each RNA. Using axolotl nuclear extracts from stage VI oocytes, we further produced evidence of destabilizing factors targeting the Awnt-5B RNAs. Altogether, these results show that oocyte maturation and late cleavages following MBT are two important periods when axolotl Wnt RNAs are highly regulated.

Evidence for an RNA polymerization activity in axolotl and Xenopus egg extracts.

We have previously reported a post-transcriptional RNA amplification observed in vivo following injection of in vitro synthesized transcripts into axolotl oocytes, unfertilized (UFE) or fertilized eggs. To further characterize this phenomenon, low speed extracts (LSE) from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i) the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii) the phenomenon is resistant to α-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3'dAMP), but sensitive to cordycepin 5'-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii) the RNA polymerization is not a 3' end labelling and that iv) the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp).

Characterization and expression of a maternal axolotl cyclin B1 during oogenesis and early development.

The M phase promoting factor (MPF) is a dimer composed of a catalytic Cdk1 subunit and a Cyclin B regulatory subunit. We have characterized a cDNA containing the entire coding sequence of an axolotl Cyclin B1 protein that is able to promote MPF activity when added to a fraction from prophase I oocytes that contains monomeric Cdk1. The axolotl cyclin B1 gene is expressed as a maternal mRNA in oocytes and early embryos. Its poly(A) tail length increases in metaphase II oocytes and then decreases regularly during the first embryonic cell cycles. Endogenous Cyclin B1 protein is first expressed during oocyte meiotic maturation. Its level oscillates after fertilization and is coordinated to the phosphorylation level of tyrosine 15 residue of Cdk1 (pTyr15), with both maxima preceding each cell division. As expected, when translated into microinjected oocytes, axolotl Cyclin B1 induces the resumption of meiosis. In electrically activated unfertilized eggs (UFE), Cyclin B1 and pTyr15 cyclic accumulations are observed with kinetics different from those of the early embryonic cycles. The axolotl embryo and UFE provide interesting in vivo comparative models for studying events controlling Cyclin B1 regulation during development.

Coupled amplification and degradation of exogenous RNA injected in amphibian oocytes.

The early development of amphibians takes place in the absence of significant transcription and is controlled at the post-transcriptional level. We have reported that in vitro synthesized transcripts injected into axolotl fertilized eggs or oocytes were not continuously degraded as their abundance apparently fluctuated over time, with detected amounts sometimes higher than initial injected amounts. To further characterize this phenomenon, we have co-injected RNA chain terminators to prevent RNA synthesis. This led to the suppression of fluctuations and to a regular decrease in the amount of transcripts that appeared to be more stable in the presence of inhibitors. These observations indicate a coupling between RNA synthesis and an accelerated degradation. Throughout the time course, cRNA molecules could be detected, and their abundance increased in the early phase of the kinetics, supporting the implication of an RNA-dependent RNA polymerase in an asymmetric amplification process. Finally, when the fate of the injected transcripts was investigated in individual oocytes, we observed an absolute increase in abundance in some but not all oocytes, supporting the existence of a limiting step in the initiation of the RNA amplification stochastic process.

Activation of Cdc2 kinase during meiotic maturation of axolotl oocyte.

Activity of Cdc2, the universal inducer of mitosis, is regulated by phosphorylation and binding to cyclin B. Comparative studies using oocytes from several amphibian species have shown that different mechanisms allow Cdc2 activation and entry into first meiotic division. In Xenopus, immature oocytes stockpile pre-M-phase promoting factor (MPF) composed of Cdc2-cyclin B complexes maintained inactive by Thr14 and Tyr15 phosphorylation of Cdc2. Activation of MPF relies on the conversion of pre-MPF into MPF by Cdc2 dephosphorylation, implying a positive feedback loop known as MPF auto-amplification. On the contrary, it has been proposed that pre-MPF is absent in immature oocyte and that MPF activation depends on cyclin synthesis in some fishes and other amphibians. We demonstrate here that MPF activation in the axolotl oocyte, an urodele amphibian, is achieved through mechanisms resembling partly those found in Xenopus oocyte. Pre-MPF is present in axolotl immature oocyte and is activated during meiotic maturation. However, monomeric Cdc2 is expressed in large excess over pre-MPF, and pre-MPF activation by Cdc2 dephosphorylation takes place progressively and not abruptly as in Xenopus oocyte. The intracellular compartmentalization as well as the low level of pre-MPF in axolotl oocyte could account for the differences in oocyte MPF activation in both species.

Injection of exogenous RNA in amphibian oocytes leads to RNA level fluctuations which are sensitive to cordycepin, an RNA chain elongation terminator.

Using an in vivo heterologous system to study the stability of Xenopus laevis RNA injected into axolotl (Ambystoma mexicanum) fertilized eggs, we have previously observed unexpected fluctuations in RNA level during early development [Andéol et al., Differentiation 63 (1998) 69-79]. In this study, we further characterize this phenomenon and establish its existence during axolotl and Xenopus oogenesis, suggesting a phylogenetically conserved mechanism. The phenomenon can occur with a variety of exogenous sense and antisense substrates. RNase protection experiments establish that most of the molecules have the same polarity as the initially injected RNA. In addition, trace amount of complementary RNA (cRNA) can be detected the injected samples. Cordycepin prevent increases in RNA levels indicating the involvement of an RNA synthesis. These results indicate the existence of an in vivo post-transcriptional RNA amplification mechanism during the early development of amphibians.

Differential post-transcriptional regulations of wnt mRNAs upon axolotl meiotic maturation.

The products of the Wntgene family play an essential role in several aspects of embryo patterning. We have investigated the post-transcriptional regulation of three of these genes: Awnt-1, Awnt-5A and Awnt-5B during axolotl (Ambystoma mexicanum) oogenesis, oocyte maturation and early development. We show that Awnt-1, Awnt-5A and Awnt-5B mRNAs are maternally expressed. The three transcripts are tightly regulated at specific times and display differential mRNA stability, poly(A) tail length and localization. In contrastto Awnt-5Bwhich is restricted to the animal hemisphere, Awnt-1 and Awnt-5A have no particular localization in stage VI oocytes. Interestingly, these two mRNAs exhibit a polyadenylation gradient along the animal-vegetal axis. Moreover, after meiotic maturation, Awnt-1 and 5A mRNAs become exclusively localized to the animal pole. This isthe first evidence of a complete mRNA re-localization to the animal hemisphere during oocyte maturation.

Isolation and developmental expression of an oogenesis-specific Xenopus cDNA clone.

We have characterized a cDNA clone designated XSYO, complementary to a transcript that is highly expressed during Xenopus oogenesis. XSYO is expressed as a maternal mRNA in oocytes and early embryos at a level up to 3 × 108 copies per mature oocyte. This level is 100-fold higher than the concentration of an average maternal RNA in the oocyte and close to the value found for the stockpiled maternal histone mRNA. This level remains constant thoughout the first rapid cleavage stages until the midblastula transition (MBT). After this stage, the XSYO RNA is degraded within 2 h and, after the blastula stage, a very weak expression was detected. This gene is not expressed in Xenopus proliferative somatic cultured cells, suggesting that it is not a simple housekeeping gene. The presence of a potential metal-binding domain in the XSYO sequence suggests that this gene might code for a protein involved in nucleic acid binding or gene regulation specific to oogenesis or early development.