The conserved regulatory basis of mRNA contributions to the early Drosophila embryo differs between the maternal and zygotic genomes.

The gene products that drive early development are critical for setting up developmental trajectories in all animals. The earliest stages of development are fueled by maternally provided mRNAs until the zygote can take over transcription of its own genome. In early development, both maternally deposited and zygotically transcribed gene products have been well characterized in model systems. Previously, we demonstrated that across the genus Drosophila, maternal and zygotic mRNAs are largely conserved but also showed a surprising amount of change across species, with more differences evolving at the zygotic stage than the maternal stage. In this study, we use comparative methods to elucidate the regulatory mechanisms underlying maternal deposition and zygotic transcription across species. Through motif analysis, we discovered considerable conservation of regulatory mechanisms associated with maternal transcription, as compared to zygotic transcription. We also found that the regulatory mechanisms active in the maternal and zygotic genomes are quite different. For maternally deposited genes, we uncovered many signals that are consistent with transcriptional regulation at the level of chromatin state through factors enriched in the ovary, rather than precisely controlled gene-specific factors. For genes expressed only by the zygotic genome, we found evidence for previously identified regulators such as Zelda and GAGA-factor, with multiple analyses pointing toward gene-specific regulation. The observed mechanisms of regulation are consistent with what is known about regulation in these two genomes: during oogenesis, the maternal genome is optimized to quickly produce a large volume of transcripts to provide to the oocyte; after zygotic genome activation, mechanisms are employed to activate transcription of specific genes in a spatiotemporally precise manner. Thus the genetic architecture of the maternal and zygotic genomes, and the specific requirements for the transcripts present at each stage of embryogenesis, determine the regulatory mechanisms responsible for transcripts present at these stages.

Natural variation in the maternal and zygotic mRNA complements of the early embryo in Drosophila melanogaster.

BACKGROUND: Maternal gene products supplied to the egg during oogenesis drive the earliest events of development in all metazoans. After the initial stages of embryogenesis, maternal transcripts are degraded as zygotic transcription is activated; this is known as the maternal to zygotic transition (MZT). Recently, it has been shown that the expression of maternal and zygotic transcripts have evolved in the Drosophila genus over the course of 50 million years. However, the extent of natural variation of maternal and zygotic transcripts within a species has yet to be determined. We asked how the maternal and zygotic pools of mRNA vary within and between populations of D. melanogaster. In order to maximize sampling of genetic diversity, African lines of D. melanogaster originating from Zambia as well as DGRP lines originating from North America were chosen for transcriptomic analysis. RESULTS: Generally, we find that maternal transcripts are more highly conserved, and zygotic transcripts evolve at a higher rate. We find that there is more within-population variation in transcript abundance than between populations and that expression variation is highest post- MZT between African lines. CONCLUSIONS: Determining the natural variation of gene expression surrounding the MZT in natural populations of D. melanogaster gives insight into the extent of how a tightly regulated process may vary within a species, the extent of developmental constraint at both stages and on both the maternal and zygotic genomes, and reveals expression changes allowing this species to adapt as it spread across the world.

The early embryonic transcriptome of a Hawaiian Drosophila picture-wing fly shows evidence of altered gene expression and novel gene evolution.

A massive adaptive radiation on the Hawaiian archipelago has produced approximately one-quarter of the fly species in the family Drosophilidae. The Hawaiian Drosophila clade has long been recognized as a model system for the study of both the ecology of island endemics and the evolution of developmental mechanisms, but relatively few genomic and transcriptomic datasets are available for this group. We present here a differential expression analysis of the transcriptional profiles of two highly conserved embryonic stages in the Hawaiian picture-wing fly Drosophila grimshawi. When we compared our results to previously published datasets across the family Drosophilidae, we identified cases of both gains and losses of gene representation in D. grimshawi, including an apparent delay in Hox gene activation. We also found a high expression of unannotated genes. Most transcripts of unannotated genes with open reading frames do not have identified homologs in non-Hawaiian Drosophila species, although the vast majority have sequence matches in genomes of other Hawaiian picture-wing flies. Some of these unannotated genes may have arisen from noncoding sequence in the ancestor of Hawaiian flies or during the evolution of the clade. Our results suggest that both the modified use of ancestral genes and the evolution of new ones may occur in rapid radiations.

Evolution of maternal and zygotic mRNA complements in the early Drosophila embryo.

The earliest stages of animal development are controlled by maternally deposited mRNA transcripts and proteins. Once the zygote is able to transcribe its own genome, maternal transcripts are degraded, in a tightly regulated process known as the maternal to zygotic transition (MZT). While this process has been well-studied within model species, we have little knowledge of how the pools of maternal and zygotic transcripts evolve. To characterize the evolutionary dynamics and functional constraints on early embryonic expression, we created a transcriptomic dataset for 14 Drosophila species spanning over 50 million years of evolution, at developmental stages before and after the MZT, and compared our results with a previously published Aedes aegypti developmental time course. We found deep conservation over 250 million years of a core set of genes transcribed only by the zygote. This select group is highly enriched in transcription factors that play critical roles in early development. However, we also identify a surprisingly high level of change in the transcripts represented at both stages over the phylogeny. While mRNA levels of genes with maternally deposited transcripts are more highly conserved than zygotic genes, those maternal transcripts that are completely degraded at the MZT vary dramatically between species. We also show that hundreds of genes have different isoform usage between the maternal and zygotic genomes. Our work suggests that maternal transcript deposition and early zygotic transcription are remarkably dynamic over evolutionary time, despite the widespread conservation of early developmental processes.

Sex Bias and Maternal Contribution to Gene Expression Divergence in Drosophila Blastoderm Embryos.

Early embryogenesis is a unique developmental stage where genetic control of development is handed off from mother to zygote. Yet the contribution of this transition to the evolution of gene expression is poorly understood. Here we study two aspects of gene expression specific to early embryogenesis in Drosophila: sex-biased gene expression prior to the onset of canonical X chromosomal dosage compensation, and the contribution of maternally supplied mRNAs. We sequenced mRNAs from individual unfertilized eggs and precisely staged and sexed blastoderm embryos, and compared levels between D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis. First, we find that mRNA content is highly conserved for a given stage and that studies relying on pooled embryos likely systematically overstate the degree of gene expression divergence. Unlike studies done on larvae and adults where most species show a larger proportion of genes with male-biased expression, we find that transcripts in Drosophila embryos are largely female-biased in all species, likely due to incomplete dosage compensation prior to the activation of the canonical dosage compensation mechanism. The divergence of sex-biased gene expression across species is observed to be often due to lineage-specific decrease of expression; the most drastic example of which is the overall reduction of male expression from the neo-X chromosome in D. pseudoobscura, leading to a pervasive female-bias on this chromosome. We see no evidence for a faster evolution of expression on the X chromosome in embryos (no "faster-X" effect), unlike in adults, and contrary to a previous study on pooled non-sexed embryos. Finally, we find that most genes are conserved in regard to their maternal or zygotic origin of transcription, and present evidence that differences in maternal contribution to the blastoderm transcript pool may be due to species-specific divergence of transcript degradation rates.

Sex-specific embryonic gene expression in species with newly evolved sex chromosomes.

Sex chromosome dosage differences between females and males are a significant form of natural genetic variation in many species. Like many species with chromosomal sex determination, Drosophila females have two X chromosomes, while males have one X and one Y. Fusions of sex chromosomes with autosomes have occurred along the lineage leading to D. pseudoobscura and D. miranda. The resulting neo-sex chromosomes are gradually evolving the properties of sex chromosomes, and neo-X chromosomes are becoming targets for the molecular mechanisms that compensate for differences in X chromosome dose between sexes. We have previously shown that D. melanogaster possess at least two dosage compensation mechanisms: the well- characterized MSL-mediated dosage compensation active in most somatic tissues, and another system active during early embryogenesis prior to the onset of MSL-mediated dosage compensation. To better understand the developmental constraints on sex chromosome gene expression and evolution, we sequenced mRNA from individual male and female embryos of D. pseudoobscura and D. miranda, from ∼0.5 to 8 hours of development. Autosomal expression levels are highly conserved between these species. But, unlike D. melanogaster, we observe a general lack of dosage compensation in D. pseudoobscura and D. miranda prior to the onset of MSL-mediated dosage compensation. Thus, either there has been a lineage-specific gain or loss in early dosage compensation mechanism(s) or increasing X chromosome dose may strain dosage compensation systems and make them less effective. The extent of female bias on the X chromosomes decreases through developmental time with the establishment of MSL-mediated dosage compensation, but may do so more slowly in D. miranda than D. pseudoobscura. These results also prompt a number of questions about whether species with more sex-linked genes have more sex-specific phenotypes, and how much transcript level variance is tolerable during critical stages of development.

An essential role for zygotic expression in the pre-cellular Drosophila embryo.

The Drosophila embryo proceeds through thirteen mitotic divisions as a syncytium. Its nuclei distribute in the embryo's interior during the first six divisions, dividing synchronously with a cycle time of less than ten minutes. After seven divisions (nuclear cycle 8), the syncytial blastoderm forms as the nuclei approach the embryo surface and slow their cycle time; subsequent divisions proceed in waves that initiate at the poles. Because genetic studies have not identified zygotic mutants that affect the early divisions and because transcription has not been detected before cycle 8, the early, pre-blastoderm embryo has been considered to rely entirely on maternal contributions and to be transcriptionally silent. Our studies identified several abnormal phenotypes in live engrailed (en) mutant embryos prior to cycle 8, as well as a small group of genes that are transcribed in embryos prior to cycle 7. Nuclei in en embryos divide asynchronously, an abnormality that was detected as early as nuclear cycle 2-3. Anti-En antibody detected nuclear En protein in embryos at cycle 2, and expression of an En:GFP fusion protein encoded in the paternal genome was also detected in cycle 2 nuclei. These findings demonstrate that the Drosophila embryo is functionally competent for gene expression prior to the onset of its rapid nuclear divisions and that the embryo requires functions that are expressed in the zygote in order to faithfully prosecute its early, pre-cellularization mitotic cycles.

Extensive divergence of transcription factor binding in Drosophila embryos with highly conserved gene expression.

To better characterize how variation in regulatory sequences drives divergence in gene expression, we undertook a systematic study of transcription factor binding and gene expression in blastoderm embryos of four species, which sample much of the diversity in the 40 million-year old genus Drosophila: D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis. We compared gene expression, measured by mRNA-seq, to the genome-wide binding, measured by ChIP-seq, of four transcription factors involved in early anterior-posterior patterning. We found that mRNA levels are much better conserved than individual transcription factor binding events, and that changes in a gene's expression were poorly explained by changes in adjacent transcription factor binding. However, highly bound sites, sites in regions bound by multiple factors and sites near genes are conserved more frequently than other binding, suggesting that a considerable amount of transcription factor binding is weakly or non-functional and not subject to purifying selection.

Noncanonical compensation of zygotic X transcription in early Drosophila melanogaster development revealed through single-embryo RNA-seq.

When Drosophila melanogaster embryos initiate zygotic transcription around mitotic cycle 10, the dose-sensitive expression of specialized genes on the X chromosome triggers a sex-determination cascade that, among other things, compensates for differences in sex chromosome dose by hypertranscribing the single X chromosome in males. However, there is an approximately 1 hour delay between the onset of zygotic transcription and the establishment of canonical dosage compensation near the end of mitotic cycle 14. During this time, zygotic transcription drives segmentation, cellularization, and other important developmental events. Since many of the genes involved in these processes are on the X chromosome, we wondered whether they are transcribed at higher levels in females and whether this might lead to sex-specific early embryonic patterning. To investigate this possibility, we developed methods to precisely stage, sex, and characterize the transcriptomes of individual embryos. We measured genome-wide mRNA abundance in male and female embryos at eight timepoints, spanning mitotic cycle 10 through late cycle 14, using polymorphisms between parental lines to distinguish maternal and zygotic transcription. We found limited sex-specific zygotic transcription, with a weak tendency for genes on the X to be expressed at higher levels in females. However, transcripts derived from the single X chromosome in males were more abundant that those derived from either X chromosome in females, demonstrating that there is widespread dosage compensation prior to the activation of the canonical MSL-mediated dosage compensation system. Crucially, this new system of early zygotic dosage compensation results in nearly identical transcript levels for key X-linked developmental regulators, including giant (gt), brinker (brk), buttonhead (btd), and short gastrulation (sog), in male and female embryos.

Canalization of segmentation and its evolution in Drosophila.

Segmentation in Drosophila embryogenesis occurs through a hierarchical cascade of regulatory gene expression driven by the establishment of a diffusion-mediated morphogen gradient. Here, we investigate the response of this pattern formation process to genetic variation and evolution in egg size. Specifically, we ask whether spatial localization of gap genes Kruppel (Kr) and giant (gt) and the pair-rule gene even-skipped (eve) during cellularization is robust to genetic variation in embryo length in three Drosophila melanogaster isolines and two closely related species. We identified two wild-derived strains of D. melanogaster whose eggs differ by approximately 25% in length when reared under identical conditions. These two lines, a D. melanogaster laboratory stock (w1118), and offspring from crosses between the lines all exhibit precise scaling in the placement of gap and pair-rule gene expression along the anterior-posterior axis in relation to embryo length. Genetic analysis indicates that this scaling is maternally controlled. Maternal regulation of scaling must be required for consistent localization of segmentation gene expression because embryo size, a genetically variable and adaptive trait, is maternally inherited. We also investigated spatial scaling between these D. melanogaster lines and single lines of Drosophila sechellia and Drosophila simulans, the latter two differing by approximately 25% in egg length. In contrast to the robust scaling we observed within species, localization of gene expression relative to embryo length differs significantly between the three species. Thus, the developmental mechanism that assures robust scaling within a species does not prevent rapid evolution between species.

Evolved Differences in cis and trans Regulation Between the Maternal and Zygotic mRNA Complements in the Drosophila Embryo.

How gene expression can evolve depends on the mechanisms driving gene expression. Gene expression is controlled in different ways in different developmental stages; here we ask whether different developmental stages show different patterns of regulatory evolution. To explore the mode of regulatory evolution, we used the early stages of embryonic development controlled by two different genomes, that of the mother and that of the zygote. During embryogenesis in all animals, initial developmental processes are driven entirely by maternally provided gene products deposited into the oocyte. The zygotic genome is activated later, when developmental control is handed off from maternal gene products to the zygote during the maternal-to-zygotic transition. Using hybrid crosses between sister species of Drosophila (Dsimulans, D. sechellia, and D. mauritiana) and transcriptomics, we find that the regulation of maternal transcript deposition and zygotic transcription evolve through different mechanisms. We find that patterns of transcript level inheritance in hybrids, relative to parental species, differ between maternal and zygotic transcripts, and maternal transcript levels are more likely to be conserved. Changes in transcript levels occur predominantly through differences in trans regulation for maternal genes, while changes in zygotic transcription occur through a combination of both cis and trans regulatory changes. Differences in the underlying regulatory landscape in the mother and the zygote are likely the primary determinants for how maternal and zygotic transcripts evolve.

Evolution of larval segment position across 12 Drosophila species.

Many developmental traits that are critical to the survival of the organism are also robust. These robust traits are resistant to phenotypic change in the face of variation. This presents a challenge to evolution. In this article, we asked whether and how a well-established robust trait, Drosophila segment patterning, changed over the evolutionary history of the genus. We compared segment position scaled to body length at the first-instar larval stage among 12 Drosophila species. We found that relative segment position has changed many times across the phylogeny. Changes were frequent, but primarily small in magnitude. Phylogenetic analysis demonstrated that rates of change in segment position are variable along the Drosophila phylogenetic tree, and that these changes can occur in short evolutionary timescales. Correlation between position shifts of segments decreased as the distance between two segments increased, suggesting local control of segment position. The posterior-most abdominal segment showed the highest magnitude of change on average, had the highest rate of evolution between species, and appeared to be evolving more independently as compared to the rest of the segments. This segment was exceptionally elongated in the cactophilic species in our dataset, raising questions as to whether this change may be adaptive.

Effect of Larval Nutrition on Maternal mRNA Contribution to the Drosophila Egg.

Embryonic development begins under the control of maternal gene products, mRNAs and proteins that the mother deposits into the egg; the zygotic genome is activated some time later. Maternal control of early development is conserved across metazoans. Gene products contributed by mothers are critical to many early developmental processes, and set up trajectories for the rest of development. Maternal deposition of these factors is an often-overlooked aspect of parental investment. If the mother experiences challenging environmental conditions, such as poor nutrition, previous studies in Drosophila melanogaster have demonstrated a plastic response wherein these mothers may produce larger eggs to buffer the offspring against the same difficult environment. This additional investment can produce offspring that are more fit in the challenging environment. With this study, we ask whether D. melanogaster mothers who experience poor nutrition during their own development change their gene product contribution to the egg. We perform mRNA-Seq on eggs at a stage where all mRNAs are maternally derived, from mothers with different degrees of nutritional limitation. We find that nutritional limitation produces similar transcript changes at all degrees of limitation tested. Genes that have lower transcript abundance in nutritionally limited mothers are those involved in translation, which is likely one of the most energetically costly processes occurring in the early embryo. We find an increase in transcripts for transport and localization of macromolecules, and for the electron transport chain. The eggs produced by nutrition-limited mothers show a plastic response in mRNA deposition, which may better prepare the future embryo for development in a nutrition-limited environment.

Evolution and inheritance of early embryonic patterning in Drosophila simulans and D. sechellia.

Pattern formation in Drosophila is a widely studied example of a robust developmental system. Such robust systems pose a challenge to adaptive evolution, as they mask variation that selection may otherwise act upon. Yet we find variation in the localization of expression domains (henceforth "stripe allometry") in the pattern formation pathway. Specifically, we characterize differences in the gap genes giant and Kruppel, and the pair-rule gene even-skipped, which differ between the sibling species Drosophila simulans and D. sechellia. In a double-backcross experiment, stripe allometry is consistent with maternal inheritance of stripe positioning and multiple genetic factors, with a distinct genetic basis from embryo length. Embryos produced by F1 and F2 backcross mothers exhibit novel spatial patterns of gene expression relative to the parental species, with no measurable increase in positional variance among individuals. Buffering of novel spatial patterns in the backcross genotypes suggests that robustness need not be disrupted in order for the trait to evolve, and perhaps the system is incapable of evolving to prevent the expression of all genetic variation. This limitation, and the ability of natural selection to act on minute genetic differences that are within the "margin of error" for the buffering mechanism, indicates that developmentally buffered traits can evolve without disruption of robustness.

Artificial selection on egg size perturbs early pattern formation in Drosophila melanogaster.

Pattern formation in Drosophila embryogenesis has been widely investigated as a developmental and evolutionary model of robustness. To ask whether genetic variation for pattern formation is suppressed in this system, artificial selection for divergent egg size was used to challenge the scaling of even-skipped (eve) pattern formation in mitotic cycle 14 (stage 5) embryos of Drosophila melanogaster. Three-dimensional confocal imaging revealed shifts in the allometry of eve pair-rule stripes along both anterior­posterior (A­P) and dorsoventral (D­V) axes as a correlated response to egg size selection, indicating the availability of genetic variation for this buffered trait. Environmental perturbation was not required for the manifestation of this variation. The number of nuclei at the cellular blastoderm stage also changed in response to selection, with large-egg selected lines having more than 1000 additional nuclei relative to small-egg lines. This increase in nuclear number in larger eggs does not scale with egg size, however, as nuclear density is inversely correlated with egg length. Nuclear density varies along the A­P axis but does not correlate with the shift in eve stripe allometry between the selection treatments. Despite its macroevolutionary conservation, both eve stripe patterning and blastoderm cell number vary genetically both within and between closely related species.