Maternal Transcripts of Hox Genes Are Found in Oocytes of Platynereis dumerilii (Annelida, Nereididae)

Abstract

Hox genes are some of the best studied developmental control genes. In the overwhelming majority of bilateral animals, these genes are sequentially activated along the main body axis during the establishment of the ground plane, i.e., at the moment of gastrulation. Their activation is necessary for the correct differentiation of cell lines, but at the same time it reduces the level of stemness. That is why the chromatin of Hox loci in the pre-gastrulating embryo is in a bivalent state. It carries both repressive and permissive epigenetic markers at H3 histone residues, leading to transcriptional repression. There is a paradox that maternal RNAs, and in some cases the proteins of the Hox genes, are present in oocytes and preimplantation embryos in mammals. Their functions should be different from the zygotic ones and have not been studied to date. Our object is the errant annelid Platynereis dumerilii. This model is convenient for studying new functions and mechanisms of regulation of Hox genes, because it is incomparably simpler than laboratory vertebrates. Using a standard RT-PCR on cDNA template which was obtained by reverse transcription using random primers, we found that maternal transcripts of almost all Hox genes are present in unfertilized oocytes of worm. We assessed the localization of these transcripts using WMISH.

Keywords: Annelida; Hox genes; Platynereis dumerilii; maternal RNA; oogenesis.

Figure 1

RT-PCR and in situ hybridization results for ten Pdum Hox genes. (A) Results of RT-PCR and in situ hybridization with antisense Dig-probes to Hox transcripts. Schematic projection of primers onto Hox transcripts. The expected size of the PCR-fragments is indicated above the double-headed arrow. Red rectangle—homeobox; yellow rectangle—hexapeptide sequence; coding region is marked in green, 5′ (left) and 3′ (right) UTRs are marked in blue; the black arrowhead indicates the position of the intron. Scaling between amplified fragments and gene sizes is not respected in the schemes. cDNA from oocytes was obtained using random primers. L-Ladder (GeneRuler DNA Ladder Mix); RNA—genomic DNA control; 0—zero control, where water was used instead of the matrix. The white numbers indicate the annealing temperature. The additional high bands visible at the 800 bp level are not a product of amplification because they are present in the control samples that do not contain Taq-pol (Supplementary Figure S2). (B) Results of in situ hybridization with sense Dig-probes. (C) Strand-specific RT-PCR. L—Ladder, (F)—Forward primer; (R)—Reverse primer; 0—zero control. White arrows mark bands synthesized from antisense transcripts.

Figure 2

The dynamics of development of bilateral animals in the coordinates of the work of the maternal and zygotic genomes. The upper part of the illustration shows a graph of the activation of the zygotic genome, which is common to many bilaterian [23,24]. Zygotic transcripts of Hox genes (marked in green) are detected after the cleavage stage, at the moment of the onset of gastrulation. Up to this point, there is an epigenetic prohibition on their work, since they violate the state of totipotency and pluripotency. Maternal transcripts of Hox genes (marked in lilac) are present in oocytes animals from all three evolutionary lineages of Bilateria. These RNAs are degraded, but in some cases are found during cleavage (mammals and Platynereis) and are even present as proteins (red check mark; HoxB9 in mammals). Hox clusters of animals with Hox-positive oocytes are shown on the right side of the figure. Asterisks mark Hox genes whose transcripts were found in oocytes. Solid line between genes indicates physical linkage where shown. Animal photos copied from Wikipedia.