Effects of rRNA gene copy number and nucleolar variation on early development: inhibition of gastrulation in rDNA-deficient chick embryos

Because of their structural and catalytic functions during protein synthesis, the 18S, 5.8S, and 28S ribosomal RNAs (rRNAs) are essential for the support of differentiation, development, and growth. The genes encoding these RNAs are present in high copy number in all eukaryotes. Although there is evidence for the existence of variation for rRNA gene copy number within higher vertebrate species, there is little knowledge concerning the effects of such variation, especially reductions, on development and viability of homeothermic vertebrates. The main objective of this study was to determine the developmental potential of chick embryos containing defined deficiencies for rRNA gene copy number in order to assess the contribution of rDNA cluster size variation to embryonic mortality in homeothermic vertebrates. This was achieved by studying a strain of chickens containing nucleolar size polymorphisms that reflect rDNA cluster size polymorphisms. Embryos exhibiting a nucleolar phenotype of one large and one very small nucleolus (Pp) are heterozygous for a reduced rDNA cluster (+/p1) and were shown in the present study to contain about 66% of the complement of rRNA genes in normal individuals (+/+) that show two large equal-sized nucleoli (PP). The +/p1 embryos were found to develop and grow normally. Embryos exhibiting a nucleolar phenotype of two very small nucleoli (pp) are homozygous for the rDNA-deficient cluster (p1/p1) and contained about 45% of the normal rDNA complement of genes. These p1/p1 embryos were arrested in their development during early gastrulation. They exhibited a characteristic morphology consisting of a dorsal invagination that was strikingly different from the primitive streak formed in +/+ and +/p1 individuals. Cellular rRNA levels were similar among the three genotypes in morula and blastula stage embryos. However, by the gastrula stage, +/+ embryos exhibited a 50% increase in rRNA which was not observed in the rDNA-deficient embryos. In comparison, heterozygotes had 80% and p1/p1 mutants had 58% of rRNA amounts of +/+ embryos. Total nucleolar size (an indicator of rRNA gene activity) measured in cells of +/+ and +/p1 embryos, at the morula to early blastula-stages of embryogenesis, was 11% of nuclear area, whereas total nucleolar size in p1/p1 embryonic cells was 6%. Taken together, these results indicate that, to achieve differentiation beyond the blastula stage of development, adequate rRNA levels are crucial. Sufficient rRNA can be produced in Pp embryos with reduced rRNA gene copies (i.e., 66% of control), but this involves a compensatory mechanism in the wild-type gene complex.