Differential stability of Drosophila embryonic mRNAs during subsequent larval development.

The relative stabilities of specific embryonic mRNAs that persist in Drosophila melanogaster larvae were determined using an approach that combined RNA density labeling with cell-free translation. Unlike the other methods commonly used to measure the decay of individual mRNAs, the density labeling approach does not depend on the use of transcriptional inhibitors or on the measurement of precursor pool specific activities. Using this approach, we have determined that different embryonic mRNA species persist for varying periods during subsequent development, with half-lives ranging from approximately 2 to approximately 30 h. The embryonic histone mRNAs are relatively unstable; they are no longer detectable by 9 h of larval development. By 41 h of larval development, 90% of the nonhistone mRNAs assayed have decayed considerably; computerized scanning densitometry of translation products indicates that these transcripts are not decaying as members of discrete half-life classes. The persisting mRNAs that remain are very long-lived; their in vitro translation products can still be detected after 91 h of larval development. We have tentatively identified the mRNAs that encode actin, tropomyosin, and tubulin as members of this stable mRNA population. Although embryonic mRNAs do fall into these three broad classes of stability, they appear to decay with a continuum of half-lives. Because the range of half-lives is so great, mRNA stability is probably an important factor controlling mRNA abundance during Drosophila development.

A labile period in the determination of the anterior-posterior axis during early neural development in Xenopus.

The process by which the vertebrate central nervous system acquires its regional properties remains a central problem in developmental biology. It is generally argued that at early gastrula stages the dorsal mesoderm possesses precise anterior-posterior positional information, which is subsequently imparted to the overlying ectoderm. However, using regionally specific gene probes to monitor regional responses in Xenopus embryos, we find that anterior-posterior properties are not fixed until early neurula stages. During gastrulation the regional inducing capacities of the dorsal mesoderm as well as the regional responses of the presumptive neural ectoderm are activated along the entire anterior-posterior axis when these properties are assayed in recombinant and explant experiments, respectively. Restriction of regional inducing capacity in the mesoderm and responsiveness in the neural ectoderm occur only at neural plate stages.

Vertebrate eye development.

Vertebrate eye determination is mediated by a series of inductive interactions that have now been more precisely defined with the use of regional markers. Analyses of the genes responsible for eye mutations and the cloning of genes delimiting spatial domains within the developing eye have begun to elucidate the molecular basis of this process.

Embryonic lens induction: shedding light on vertebrate tissue determination.

The principle of embryonic induction was defined by early studies of lens determination, and because of the relative simplicity of the developing lens and its interaction with presumptive retinal tissue it has been a favored system for examining mechanisms of induction. Recent studies have led to substantial alterations of the classic model for this process, introducing several elements that significantly refine our view of vertebrate tissue determination.

Xenopus gamma-crystallin gene expression: evidence that the gamma-crystallin gene family is transcribed in lens and nonlens tissues.

Crystallins, the major gene products of the lens, accumulate to high levels during the differentiation of the vertebrate lens. Although crystallins were traditionally thought to be lens specific, it has recently been shown that some are also expressed at very low levels in nonlens tissues. We have examined the embryonic expression pattern of gamma-crystallins, the most abundant crystallins of the embryonic lens in Xenopus laevis. The expression profile of five Xenopus gamma-crystallin genes mirrors the pattern of lens differentiation in X. laevis, exhibiting on average a 100-fold increase between tailbud and tadpole stages. Four of these genes are also ubiquitously expressed outside the lens at a very low level, the first demonstration of nonlens expression of any gamma-crystallin gene; expression of the remaining gene was not detected outside the head region, thus suggesting that there may be two classes of gamma-crystallin genes in X. laevis. Predictions regarding control mechanisms responsible for this dual mode of expression are discussed. This study raises the question of whether any crystallin, on stringent examination, will be found exclusively in the lens.

Developmental regulation of hypomethylation of delta-crystallin genes in chicken embryo lens cells.

Sequences in the two delta-crystallin genes become hypomethylated when they are expressed in the chick lens. This system is particularly advantageous for studying temporal changes in hypomethylation, since lens tissue can be isolated at all developmental stages. In previous work we have shown that most HpaII sites become hypomethylated within the delta 1-crystallin gene long after delta-crystallin gene activation. One site is hypomethylated when crystallin mRNA begins to be synthesized at high levels at 50 h; we show here that this site maps to the 3' end (intron 15) of the delta 1-crystallin gene. In addition, we have examined the methylation status of HpaII and HhaI sites found near the 5' end of the delta 1-crystallin gene. Two HhaI sites adjacent to a viral core enhancer sequence in intron 2 are also first hypomethylated at 50 h. These findings point to regions of the delta 1 gene that should be investigated further for functional significance in regulating delta-crystallin transcription.

Genetic modulation of RNA metabolism in Drosophila. IV. Measurement of rats of RNA synthesis by density labeling.

Accumulation of RNA was measured in adult males of two genotypes: car bb/Ybb- and car bb/YbbSuVar-5. The two genotypes have similar amounts of rDNA, which is reduced in comparison to wild type (CLARK, STRAUSBAUGH and KIEFER 1977). Although genotypically bobbed, car bb/YbbSuVar-5 flies have a wild-type phenotype; car bb/Ybb- flies are both phenotypically and genotypically bobbed (CLARK, STRAUSBAUGH and KIEFER 1977). The wild-type phenotype observed in the car bb/YbbSuVar-5 flies is thought to be the result of an increased rate of rRNA synthesis due to the presence of the YbbSuVar-5 chromosome (SHERMOEN and KIFFER 1975; CLARK, STRAUSBAUGH and KIEFER 1977; CLARK and KIEFER 1977). To further define this phenomenon, the absolute accumulation of RNA was measured in the two genotypes, using density labeling methods. The accumulation of RNA is 1.4 to 1.8 times higher in car bb/YbbSuVar-5 flies than in car bb/Ybb- flies, demonstrating that there is genetic regulation of synthesis in this genotype. The use of density-labeled nucleosides has clearly shown that there is no difference in precusor pool sizes or use between the two genotypes studied.

Lens induction in axolotls: comparison with inductive signaling mechanisms in Xenopus laevis.

Amphibian lens induction is an embryonic process whose broad outlines are conserved between anurans and urodeles; however, it has been argued that some aspects of this process differ significantly between even closely related species. Classical embryologists concluded that in some species direct contact between the optic vesicle and ectoderm was both necessary and sufficient to induce the ectoderm to form a lens, while in other species tissues other than the optic vesicle induce lens formation. Recent studies of lens induction in Xenopus have argued that lens induction may be more conserved evolutionarily than was previously thought and that the different conclusions reached in the classical literature may be due more to experimental methodology than to actual differences in the process of lens induction. We have tested this hypothesis by examining the timing of lens induction in the axolotl and the ability of various tissues to induce lenses in explant cultures. We find that, despite the evolutionary divergence between Xenopus and Ambystoma, the mechanism of lens specification is substantially similar in the two species. These results support the hypothesis that the mechanism of lens induction is evolutionarily conserved among amphibians.

Characterization of Xenopus laevis gamma-crystallin-encoding genes.

In order to gain insight into crystallin (Cry)-encoding gene (cry) evolution and developmental function, we have determined the gene structure and sequence of several Xenopus laevis gamma-cry. These encode the most abundant Cry in the embryonic lens. Four of the X. laevis gamma-cry, which are part of a multigene family, were isolated from a X. laevis genomic library and demonstrated to have the same gene structure as gamma-cry from other vertebrates, thereby providing further evidence that the split between beta and gamma members of the beta gamma cry family occurred relatively early in evolution. Sequence comparisons indicate that these X. laevis genes share 88-90% nucleotide sequence identity in the protein coding regions, which is slightly higher than the identity observed between gamma-cry of other species. The 5' upstream regions of X. laevis gamma-cry contain a few short stretches of homology and one putative promoter element conserved among all cry genes but lack other regions common to gamma-cry promoters from other organisms. The deduced amino acid sequences of all four genes and one cDNA suggest that the structure of X. laevis gamma-Cry is highly conserved with that of other vertebrate gamma-Cry, as deduced from the known three-dimensional structure of bovine gamma B Cry.

Early opsin expression in Xenopus embryos precedes photoreceptor differentiation.

The visual pigment which serves as the first step in the phototransduction cycle in vertebrate rod cells consists of a retinal chromophore which is linked to the transmembrane protein, opsin. Opsin genes have been isolated from a number of different organisms and studies have shown opsin to be developmentally regulated with both mRNA and protein expression associated with the morphological differentiation of photoreceptor cells. Due to its potential utility as a marker for rod photoreceptor determination in studies of retinal tissue interactions, and because no amphibian opsin genes have as yet been cloned, we isolated cDNA clones of the Xenopus laevis opsin gene. Sequence analysis shows that within the coding region Xenopus opsin shares a high degree of identity with other rod opsin genes, except at the C-terminal where it more closely resembles the mammalian color opsins. A developmental analysis, on the other hand, reveals that Xenopus opsin transcripts are detectable in a retina-specific fashion early in retinal development. Using in situ hybridization we find that Xenopus opsin mRNA is initially restricted to a few isolated cells in the presumptive photoreceptor layer which express the gene at relatively high levels. This suggests that rod photoreceptor determination occurs in single cells, and that the mechanisms controlling opsin expression in Xenopus are initiated well before any evidence of morphological differentiation.

Changes in neural and lens competence in Xenopus ectoderm: evidence for an autonomous developmental timer.

The ability of a tissue to respond to induction, termed its competence, is often critical in determining both the timing of inductive interactions and the extent of induced tissue. We have examined the lens-forming competence of Xenopus embryonic ectoderm by transplanting it into the presumptive lens region of open neural plate stage embryos. We find that early gastrula ectoderm has little lens-forming competence, but instead forms neural tissue, despite its location outside the neural plate; we believe that the transplants are being neuralized by a signal originating in the host neural plate. This neural competence is not localized to a particular region within the ectoderm since both dorsal and ventral portions of early gastrula ectoderm show the same response. As ectoderm is taken from gastrulae of increasing age, its neural competence is gradually lost, while lens competence appears and then rapidly disappears during later gastrula stages. To determine whether these developmental changes in competence result from tissue interactions during gastrulation, or are due to autonomous changes within the ectoderm itself, ectoderm was removed from early gastrulae and cultured for various periods of time before transplantation. The loss of neural competence, and the gain and loss of lens competence, all occur in ectoderm cultured in vitro with approximately the same time course as seen in ectoderm in vitro. Thus, at least from the beginning of gastrulation onwards, changes in competence occur autonomously within ectoderm. We propose that there is a developmental timing mechanism in embryonic ectoderm that specifies a sequence of competences solely on the basis of the age of the ectoderm.