45S rDNA Repeats of Turtles and Crocodiles Harbor a Functional 5S rRNA Gene Specifically Expressed in Oocytes.

In most eukaryotic genomes, tandemly repeated copies of 5S rRNA genes are clustered outside the nucleolus organizer region (NOR), which normally encodes three other major rRNAs: 18S, 5.8S, and 28S. Our analysis of turtle rDNA sequences has revealed a 5S rDNA insertion into the NOR intergenic spacer in antisense orientation. The insertion (hereafter called NOR-5S rRNA gene) has a length of 119 bp and coexists with the canonical 5S rDNA clusters outside the NOR. Despite the ∼20% nucleotide difference between the two 5S gene sequences, their internal control regions for RNA polymerase III are similar. Using the turtle Trachemys scripta as a model species, we showed the NOR-5S rDNA specific expression in oocytes. This expression is concurrent with the NOR rDNA amplification during oocyte growth. We show that in vitellogenic oocytes, the NOR-5S rRNA prevails over the canonical 5S rRNA in the ribosomes, suggesting a role of modified ribosomes in oocyte-specific translation. The orders Testudines and Crocodilia seem to be the only taxa of vertebrates with such a peculiar rDNA organization. We speculate that the amplification of the 5S rRNA genes as a part of the NOR DNA during oogenesis provides a dosage balance between transcription of all the four ribosomal RNAs while producing a maternal pool of extra ribosomes. We further hypothesize that the NOR-5S rDNA insertion appeared in the Archelosauria clade during the Permian period and was lost later in the ancestors of Aves.

Characterization of axolotl lampbrush chromosomes by fluorescence in situ hybridization and immunostaining.

The lampbrush chromosomes (LBCs) in oocytes of the Mexican axolotl (Ambystoma mexicanum) were identified some time ago by their relative lengths and predicted centromeres, but they have never been associated completely with the mitotic karyotype, linkage maps or genome assembly. We identified 9 of the axolotl LBCs using RNAseq to identify actively transcribed genes and 13 BAC (bacterial artificial clone) probes containing pieces of active genes. Using read coverage analysis to find candidate centromere sequences, we developed a centromere probe that localizes to all 14 centromeres. Measurements of relative LBC arm lengths and polymerase III localization patterns enabled us to identify all LBCs. This study presents a relatively simple and reliable way to identify each axolotl LBC cytologically and to anchor chromosome-length sequences (from the axolotl genome assembly) to the physical LBCs by immunostaining and fluorescence in situ hybridization. Our data will facilitate a more detailed transcription analysis of individual LBC loops.

On some structural and evolutionary aspects of rDNA amplification in oogenesis of Trachemys scripta turtles.

The features of rDNA amplification have been studied in oocytes of the red-eared slider Trachemys scripta using a number of specific histochemical and cytomolecular methods. A single nucleolus in early diplotene oocytes is associated with the nucleolus organizer region (NOR). With oocyte growth, the number of nucleoli increases dramatically and reaches hundreds by the lampbrush chromosome stage (pre-vitellogenesis). RNA-polymerase I, fibrillarin, and PCNA immunodetection in the amplified nucleoli and FISH of the 5'ETS probe to the oocyte nuclear content suggest pre-rRNA and rDNA synthesis in the nucleoli at all stages studied. This implies a continuous reproduction of the nucleoli during oocyte development from early diplotene up to vitellogenesis. The data obtained offer a different way for rDNA amplification and formation of extrachromosomal nucleoli in turtle oocytes compared with the amplified nucleoli formation in amphibian and fish oocytes. In the Sauropsida clade of Archelosauria, which includes turtles, crocodiles, and birds, rDNA function is known to be suppressed in avian oogenesis during the lampbrush stage (Gaginskaya et al. in Cytogenet Genome Res 124:251-267, 2009).

Dual embryonic origin of the hyobranchial apparatus in the Mexican axolotl (Ambystoma mexicanum).

Traditionally, the cartilaginous viscerocranium of vertebrates is considered as neural crest (NC)-derived. Morphological work carried out on amphibian embryos in the first half of the XX century suggested potentially mesodermal origin for some hyobranchial elements. Since then, the embryonic sources of the hyobranchial apparatus in amphibians has not been investigated due to lack of an appropriate long-term labelling system. We performed homotopic transplantations of neural folds along with the majority of cells of the presumptive NC, and/or fragments of the head lateral plate mesoderm (LPM) from transgenic GFP+ into white embryos. In these experiments, the NC-derived GFP+ cells contributed to all hyobranchial elements, except for basibranchial 2, whereas the grafting of GFP+ head mesoderm led to a reverse labelling result. The grafting of only the most ventral part of the head LPM resulted in marking of the basibranchial 2 and the heart myocardium, implying their origin from a common mesodermal region. This is the first evidence of contribution of LPM of the head to cranial elements in any vertebrate. If compared to fish, birds, and mammals, in which all branchial skeletal elements are NC-derived, the axolotl (probably this is true for all amphibians) demonstrates an evolutionary deviation, in which the head LPM replaces NC cells in a hyobranchial element. This implies that cells of different embryonic origin may have the same developmental program, leading to the formation of identical (homologous) elements of the skeleton.