Mutations that perturb poly(A)-dependent maternal mRNA activation block the initiation of development
In Drosophila, maternal effect genes are expressed during oogenesis
that produce translationally inactive mRNAs. These mRNAs are stored regionally
in the cytoplasm of the oocyte for future translation during such processes
as oocyte maturation, fertilization and early embryonic development. Some
maternal effect genes have been found to be crucial in determining embryonic
asymmetry. Drosophila mutants exist that express the same phenotypic
characteristics as those produced by selective removal of cytoplasm from
different regions of the oocyte. These mutants helped determine that the
bicoid gene is necessary for development of anterior structures such
as the head and thorax. Similarly, nanos determines posterior structures
and toll dorsoventral. These are regulatory genes producing protein
products that regulate development through transcriptional regulation of
target genes (Browder et al., 1991; Xiuguang et al., 1996).
Marshal Lieberfarb et al. (1996) studied defects in translation of the mRNAs from these genes. They used female-sterile mutants that were defective in translation of maternal mRNAs and found two genes, cortex and grauzone to be crucial to both translation and polyadenylation of bicoid and toll mRNAs. Mutants with defective cortex genes contained bicoid mRNA identical in amount, localization and structure as that found in wild-type embryos but with shortened poly(A) tails. Embryos from homozygous cortex-deficient females showed profound deficiency in Bicoid protein expression. In contrast, cortex mutants had fully translatable nanos mRNA, because translation of this mRNA was poly(A)-independent. Mutants with defective grauzone genes showed results similar to cortex mutants. Therefore, the bicoid and toll genes form a separate class of genes relying on polyadenation for translation.
To demonstrate that polyadenation was the mechanism used in translation, Lieberfarb et al. first showed differences in poly(A) length of wild-type and cortex mutant mRNAs via a PCR poly(A) test. They determined that the bicoid mRNA acquires an elongated poly(A) tail to approx. 140 nucleotides in the wild-type, whereas the mutants had their bicoid mRNA elongated to a maximun length of approx. 80 nucleotides. This indicates that the cortex gene is required for proper polyadenylation of cytoplasmic bicoid mRNA. If translational activity is dependent on the length of poly(A) tails, then injecting cortex mutants with polyadenylated bicoid mRNA should circumvent this deficiency; Lieberbarb et al. found that it did.
Because cortex-deficient embryos produced defects other than just those produced by deficiency of Bicoid protein, the researchers speculated that the gene might be required for polyadenylation and translation of a whole class of mRNAs. The Toll protein in cortex-deficient embryos was also reduced, so they tested whether polyadenylation oftoll mRNA was also affected. As in the previous tests with bicoid mRNA, the researchers found that wild-type embryos elongate toll mRNA to approx. 250 A residues, whereas cortex mutants had only about 150 poly(A)s. This confirmed their suspicion that the cortex gene is required both for proper polyadenylation and translation of multiple maternal mRNAs.
To demonstrate that mutants in cortex and grauzone genes did not have a translational defect unrelated to polyadenylation, they tested whether nanos mRNA (located in the posterior pole of the embryo and not polyadenylated) could be translated. Their results showed that production of the Nanos protein was the same in the mutants as in the wild-type.
The researchers also linked the cortex gene to defects in completion of meiosis. They examined the number of nuclei and the microtubule organization in mutant embryos and found a complete absence of microtubule organization in mutant embryos and found a complete absence of microtubule-containing spindles in the central regions. This indicates that initiation of mitotic divisions was hindered. Furthermore, no polar bodies were detected in the mutant embryos, indicating that meiosis was halted. They felt that the mechanisms hindering these processes could well be related to polyadenylation and translation.
To determine whether a developmental defect was occurring due to somatic mutations in embryos, rather than translational impairment of an entire class of mRNAs, the researchers speculated that the cortex gene would be necessary in germ cells if the latter case prevailed. In experiments with females homozygous for a deficiency in the cortex gene of only the germ cells, offspring had the same morphological features as demonstrated previously in the cortex mutant phenotype. These results indicated that a functional cortex gene is required in the germ line for polyadenylation and mRNA translation. Temperature-sensitive mutants of females or embryos showed that translation of this gene is required in mid- to late oogenesis and early embryogenesis. Shifting of the females or embryos to non-permissive temperatures at various times produced various developmental deficiencies.
The results of these studies support the conclusion that mutations in the cortex and grauzone genes disturb the regulation of polyadenylation of maternal mRNAs. When defective polyadenylation of maternal mRNAs results in shortened poly(A) mRNAs, translation cannot occur, resulting in deficiencies in oocytes of microtubule reorganization and meiosis, and deficiencies in embryonic development such as body symmetry.
|The Foundations of Developmental Biology||The Developmental Biology Journal Club|
Browder, L.W., Erickson, C.A and Jeffery, W.R. 1991. Developmental Biology.
Third edition. Saunders College Pub. Philadelphia (see pp. 97-98, 597-598).
Lieberfarb, M. E., Chu, T., Wreden, C., Theurkauf, W., Gergen, J.P. and Strickland, S. 1996. Mutations that perturb poly(A)-dependent maternal mRNA activation block the initiation of development. Development 122: 579-588.
Xiuguang, M., Yuan, D., Diepold, K., Scarborough, T. and Ma, J. 1996. The Drosophila morphogenetic protein Bicoid binds DNA cooperatively. Development 122: 1195-1206 (1996).
Copyright © 1996 Fabiola Aparicio, Doris Cheng, Jason Wilshusen,
and Sharon Clark.This material may be reproduced for educational purposes
only provided credit is given to the original source.
November 3, 1996