Till now the transcription factor Xvent-2 has been studied in Xenopus embryos only by the mRNA testing. We use immunochemical methods for testing of the Xvent-2 protein and gradient-centrifugation methods for estimation of activity of its mRNA. Our results show that the Xvent-2 protein is present in eggs and early embryos. The Xvent-2 mRNA is absent at any of these developmental stages. The majority of mRNA synthesized on the zygotic genome was stored in informosomes, while only its small part could be revealed in polysomes. The spatial patterning of the Xvent-2 protein at different developmental stages did not entirely agree with that of its mRNA. These data indicate that the Xvent-2 protein functioning in Xenopu embryos is regulated not only at the transcription, but at translation and posttranslation as well. We propose that the activation of translation on the masked Xvent-2 mRNA may lead to blood differentiation and cell migration.
Usually an expression of a gene implies the synthesis of corresponding mRNA. There is a tacit consent that an absence or a presence of a certain mRNA suggests an absence or a presence of the protein. However there are lots of evidences that mRNA may present in a cell in inactive ribonucleoprotein complexes, called by different names, from informosomes to p-bodies [
Four closely identical mRNAs (Xvent-2, Xom, Vox and Xbr) have been found in X. laevis embryos at gastrula and neurula stages [3-6]. At later stages, these mRNAs are detectable in the developing eye, tail bud, somites, bronchial arch, and about proctodeum. The mRNAs are encoded by the related genes: Xvent-2B, Xbr-1b/ Vox1, Xvent-2/Xbr-1a, Vox15, and Xom [
The spatial patterning of the Xvent-2 mRNA in Xenopus embryos is closely studied [3-6]. The specific Ab to the Xvent-2 protein enabled us to determine spatial and temporal patterning of this protein in early embryos. We show that the Xvent-2 protein is stored in eggs when its mRNA is absent. This protein is revealed at all studied developmental stages. The Xvent-2 mRNA synthesis started and grew from midblastula on, reached maximum at neurula and then it was slowly vanishing by stages 39 - 40. The major part of the Xvent-2 mRNA is masked in informosomes and is not translated in the embryos. Spatial patterning of the Xvent-2 protein and its mRNA in Xenopus laevis embryos coincide only partially. The most coincidence is revealed at neurula. The spatial patterning permits us to propose that the activation of translation on the masked Xvent-2 mRNA may lead to blood differentiation and cell migration.
X. laevis embryos were obtained, cytoplasmic extracts prepared, and centrifugation in CsCl or sucrose density gradients were performed as described previously [
The stages of the Xenopus development were determined according to the tables by [
Expression and isolation of reXvent-2 protein were described previously [
The reXvent-2 protein was concentrated by methanol precipitation, dissolved in phosphate buffer and used to raise polyclonal antiserum in rabbits [
Proteins were electrophoresed in SDS-PAAG, transferred onto nitrocellulose membrane and stained with 0.1% Ponceaus S to control the transfer. Then the membrane was washed in water and treated consequently with blocking solution (5% milk), rabbit anti-reXvent-2 serum (1:1000) and goat anti-rabbit Ab conjugated to horseradish peroxidase p-SAR Iss (IMTEK, Moscow) (1:20000) as it was proposed by Promega. The peroxidase substrate TMB (tetramethilbenzydine) (“BioTestSystems” Moscow) served for staining.
Embryos were fixed according to Klymkowsky lab manual [
In the absence of Ab we found no staining of embryos with TMB. We used some controls to avoid any nonspecific Ab staining. Control 1: immunostaining with conjugate of anti-human-immunoglobulins rabbit Ab with peroxidase. Control 2: immunostaining with the conjugate of rabbit antiXvent-2 Ab with peroxidase depleted with the reXvent-2 protein. 2ml of the working solution of the conjugated anti-Xvent-2 Ab were sequentially incubated with three pieces of nitrocellulose membrane 3 × 3 cm carrying 30 - 60 mcg of the reXvent-2 protein each. The third membrane after appropriate washing could hardly be stained with peroxidase substrate and thus we considered these anti-reXvent-2 Ab to be depleted with the reXvent-2 protein.
The anti-Xvent-2 immune rabbit serum was characterized in our earlier paper [
The PAGE and immunoblotting of protein fractions obtained from X. laevis embryos at different developmental stages from eggs to tadpoles are shown in
bility of X. laevis Xvent-2 was slightly higher than that of the recombinant protein reXvent-2 owing to the lack of a polyhistidine tag and corresponded to a protein of about 45 kDa. The calculated molecular mass for the Xvent-2 is 36.5 kDa, but some proteins, for example transcription factor Ybox1 [
Intensive nonspecific staining of embryos was revealed in our preliminary experiments where the standard procedure with primary and secondary Ab was performed. That is why in the sequel we used the affine-isolated rabbit anti-Xvent2 Ab conjugated to the horseradish peroxidase. A normal rabbit serum diluted 1:5 in TBS served as a blocking solution and as a solution for anti-Xvent-2 Ab to exclude any nonspecificity.
The results of the whole-mount immunostaining of embryos at the stages of 16 - 32 blastomers and blastula are presented in
The expression of many genes in embryo development of eukaryotes is regulated at the translational level by variety of mechanisms [
somes at all the developmental stages examined (from 9 to 17.5), and only its minor fraction was associated with polysomes [
An animal pole-derived ectoderm plays a critical role in blood cells formation [
At the neurula and early tailbud stages (
immunostaining reveals Xvent-2 along the edge of the neural tube, the presumptive brain and eyes, in the bronchial arc, in the otic vesicle, in the somites and the cement gland. The staining of the cement gland may be an artifact, as an occasional nonspecific staining of it was found earlier [
Results of other authors on the in situ hybridization at the neurula and early tailbud stages revealed that the Xvent-2 mRNA patterns were generally overlapped with those of the protein. The mRNA was found along the edge of the neural tube [3,5,6,9] along the edge of the presumptive forebrain [
At the stages 30-35 the Xvent-2 mRNA was seen in three areas: in the tail tip, the dorsal parts of eyecups and in the bronchial arch [3,5,6,9].
Our study revealed the Xvent-2 protein at stage 35 in both anterior and posterior red blood island [24,26], in the lymphatic caudal dorsal vessel, in the tail fin and around the eyes, the bronchial arches and the proctodeum (
It was shown earlier [
mRNA and lack of the protein in a tail tip confirm it. We propose, that as soon as a cell (under any signal) starts to synthesize the Xvent-2 protein, it moves towards a developing lymphatic or blood vessel. That is why Xvent-2 mRNA had not been detected either in globins-containing ventral blood island or in lymphatic or blood vessels. We propose that a possible function of the Xvent-2 protein may be not only the dorsoventral axis formation in the early embryo, but also participating in the blood and lymph development. That is why the cells which synthesize the Xvent-2 protein can be revealed along the edge of some presumptive organs at neurula. They may participate in creation of the blood system of these organs.
Our results corroborate our hypothesis on the role of activation of some mRNAs in cell transition from a competence to the determination at an embryogenesis [
A pattern of mRNA cannot sometimes give a perfect reflection of a corresponding protein pattern because of the existence of inactive mRNAs or long-living proteins. It also may be a case when after activation of some protein synthesis, cells migrate and the protein appears at another place. Thus one must investigate the pattern of gene expression not only with in situ hybridization but with immunostaining of the protein too. For transcription factor Xvent-2 we showed, that it was stored in eggs and in animal blastomers when its mRNA was absent. The major of the Xvent-2 mRNA synthesized after midblastula transition was not translated in the embryos. The spatial patterning permits us to propose that the activation of translation on the masked Xvent-2 mRNA may lead to the blood differentiation and cell migration.