Expression of of distinct maternal and somatic 5.8S, 18S and 28S rRNA types during zebrafish development

Animal science

Immunoglobulin switch-like recombination regions implicated in the formation of extrachromosomal circular 45S rDNA involved in the maternal-specific translation system of zebrafish

Cellular translation is essential to all life on earth and in recent years we have reported on the discovery of a unique dual translation system in zebrafish. In this system, a maternal-type variant shows absolute expression in eggs and is progressively replaced during embryogenesis by a somatic-type variant. There are several translation system components, all with a non-coding RNA part, that show this dual characteristic: snRNA, snoRNA, rRNA, RNaseP, tRNA, and SRP-RNA. To produce sufficient ribosomes during oogenesis, zebrafish amplify their 45S locus (18S-5.8S-28S tandem repeat) by means of extrachromosomal circular DNA (eccDNA) organized in extrachromosomal rDNA circles (ERCs). Although this cellular process is discovered quite some time ago, still little is known about the mechanisms involved. Yet, because only the 45S maternal-type (45S-M) rRNA is expressed during oogenesis, the zebrafish genome provides a rare opportunity to compare an ERC 45S locus to a non-ERC 45S locus. In this study, we analyzed the genomic composition of the 45S-M and 45S-S (somatic-type) loci in combination with ultra-long read Nanopore sequencing of ERCs present in total DNA isolated from zebrafish eggs. We discovered 45S-M flanking sequences that were absent in the 45S-S locus and showed high homology to immunoglobulin (Ig) switch regions. Also, several other unique G-quadruplex DNA containing regions were found in the 45S-M locus. Some of those auxiliary regions showed different sizes in the sequenced ERCs, although within each ERC they appear to have identical sizes. These results point to a two-step system for ERC synthesis in zebrafish oogenesis: first the 45S-M repeat is excised from the chromosome into an ERC by recombination that uses the flanking Ig switch-like regions, after which the initial ECR is multiplied and extended into many ECRs with a varying number of 45S-M repeats

Cellular Factors Involved in Transcriptome Dynamics in Early Zebrafish Embryogenesis

At gastrulation in the zebrafish embryogenesis, the embryonic genome is switched on to produce transcripts that are used for the maintenance and development of the embryo. In a previous study from late blastula to mid gastrula on the transcriptomes of 179 individual embryos, we capture the transcriptome dynamics via ten gene-expression types. Here we study the factors that regulate these transcriptome dynamics by in extensive silico analyses and two small-RNA sequencing experiments. We analyzed mechanisms that would make it possible for the embryo to achieve the tight regulation of gene expression that was observed, not only during development, but also when individual embryos were compared. We found that many of the gene-expression regulatory factors that are available to the embryo are operational in the different gene-expression types and act concurrently with not one mechanism prevailing in this developmental phase. We also saw that at least one of the regulatory mechanisms, the expression of members of the miRNA-430 family again is very tightly regulated, both during development as well as when miRNA expression from individual embryos is compared

Mother-Specific Signature in the Maternal Transcriptome Composition of Mature, Unfertilized Zebrafish Eggs

Maternal mRNA present in mature oocytes plays an important role in the proper development of the early embryo. As the composition of the maternal transcriptome in general has been studied with pooled mature eggs, potential differences between individual eggs are unknown. Here we present a transcriptome study on individual zebrafish eggs from clutches of five mothers in which we focus on the differences in maternal mRNA abundance per gene between and within clutches. To minimize technical interference, we used mature, unfertilized eggs from siblings. About half of the number of analyzed genes was found to be expressed as maternal RNA. The expressed and non-expressed genes showed that maternal mRNA accumulation is a non-random process, as it is related to specific biological pathways and processes relevant in early embryogenesis. Moreover, it turned out that overall the composition of the maternal transcriptome is tightly regulated as about half of the expressed genes display a less than twofold expression range between the observed minimum and maximum expression values of a gene in the experiment. Even more, the maximum gene-expression difference within clutches is for 88% of the expressed genes lower than twofold. This means that expression differences observed in maternally expressed genes are primarily caused by differences between mothers, with only limited variability between eggs from the same mother. This was underlined by the fact that 99% of the expressed genes were found to be differentially expressed between any of the mothers in an ANOVA test. Furthermore, linking chromosome location, transcription factor binding sites, and miRNA target sites of the genes in clusters of distinct and unique mother-specific gene-expression, suggest biological relevance of the mother-specific signatures in the maternal transcriptome composition. Altogether, the maternal transcriptome composition of mature zebrafish oocytes seems to be tightly regulated with a distinct mother-specific signature

Maternal- and Somatic-type snoRNA Expression and Processing in Zebrafish Development

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that play an important role in the complex maturation process of ribosomal RNAs (rRNAs). SnoRNAs are categorized in classes, with each class member having several variants present in a genome. Similar to our finding of specific rRNA expression types in zebrafish embryogenesis, we discovered preferential maternal- and somatic-expression for snoRNAs. Most snoRNAs and their variants have higher expression levels in somatic tissues than in eggs, yet we identified three snoRNAs; U3, U8 and snoZ30 of which specific variants show maternal- or somatic-type expression. For U3 and U8 we also found small-derived snoRNAs that lack their 5’ rRNA recognition part and are essentially Domain II hairpin structures (U-DII). These U-DII snoRNAs from variants showed similar preferential expression, in which maternal-type variants are prominently expressed in eggs and subsequently replaced by a somatic-type variants during embryogenesis. This differential expression is related to the organization in tandem repeats (maternal type) or solitary (somatic-type) genes of the involved U snoRNA loci. The collective data showed convincingly that the preferential expression of snoRNAs is achieved by transcription regulation, as well as through RNA processing. Finally, we observed small-RNAs derived from internal transcribed spacers (ITSs) of a U3 snoRNA loci that via complementarity binding, may be involved in the biosynthesis of U3-DII snoRNAs. Altogether, the here described maternal- and somatic-type snoRNAs are the latest addition to the developing story about the dual ribosome system in zebrafish development