Cooperative assembly confers regulatory specificity and long-term genetic circuit stability.

A ubiquitous feature of eukaryotic transcriptional regulation is cooperative self-assembly between transcription factors (TFs) and DNA cis-regulatory motifs. It is thought that this strategy enables specific regulatory connections to be formed in gene networks between otherwise weakly interacting, low-specificity molecular components. Here, using synthetic gene circuits constructed in yeast, we find that high regulatory specificity can emerge from cooperative, multivalent interactions among artificial zinc-finger-based TFs. We show that circuits "wired" using the strategy of cooperative TF assembly are effectively insulated from aberrant misregulation of the host cell genome. As we demonstrate in experiments and mathematical models, this mechanism is sufficient to rescue circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our naturally inspired approach offers a simple, generalizable means for building high-fidelity, evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.

A Mutation in the Drosophila melanogaster eve Stripe 2 Minimal Enhancer Is Buffered by Flanking Sequences.

Enhancers are DNA sequences composed of transcription factor binding sites that drive complex patterns of gene expression in space and time. Until recently, studying enhancers in their genomic context was technically challenging. Therefore, minimal enhancers, the shortest pieces of DNA that can drive an expression pattern that resembles a gene's endogenous pattern, are often used to study features of enhancer function. However, evidence suggests that some enhancers require sequences outside the minimal enhancer to maintain function under environmental perturbations. We hypothesized that these additional sequences also prevent misexpression caused by a transcription factor binding site mutation within a minimal enhancer. Using the Drosophila melanogastereven-skipped stripe 2 enhancer as a case study, we tested the effect of a Giant binding site mutation (gt-2) on the expression patterns driven by minimal and extended enhancer reporter constructs. We found that, in contrast to the misexpression caused by the gt-2 binding site deletion in the minimal enhancer, the same gt-2 binding site deletion in the extended enhancer did not have an effect on expression. The buffering of expression levels, but not expression pattern, is partially explained by an additional Giant binding site outside the minimal enhancer. Deleting the gt-2 binding site in the endogenous locus had no significant effect on stripe 2 expression. Our results indicate that rules derived from mutating enhancer reporter constructs may not represent what occurs in the endogenous context.

Quantitative Comparison of the Anterior-Posterior Patterning System in the Embryos of Five Drosophila Species.

Complex spatiotemporal gene expression patterns direct the development of the fertilized egg into an adult animal. Comparisons across species show that, in spite of changes in the underlying regulatory DNA sequence, developmental programs can be maintained across millions of years of evolution. Reciprocally, changes in gene expression can be used to generate morphological novelty. Distinguishing between changes in regulatory DNA that lead to changes in gene expression and those that do not is therefore a central goal of evolutionary developmental biology. Quantitative, spatially-resolved measurements of developmental gene expression patterns play a crucial role in this goal, enabling the detection of subtle phenotypic differences between species and the development of computations models that link the sequence of regulatory DNA to expression patterns. Here we report the generation of two atlases of cellular resolution gene expression measurements for the primary anterior-posterior patterning genes in Drosophila simulans and Drosophila virilis By combining these data sets with existing atlases for three other Drosophila species, we detect subtle differences in the gene expression patterns and dynamics driving the highly conserved axis patterning system and delineate inter-species differences in the embryonic morphology. These data sets will be a resource for future modeling studies of the evolution of developmental gene regulatory networks.

Hunchback is counter-repressed to regulate even-skipped stripe 2 expression in Drosophila embryos.

Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. We investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped (eve). While Hunchback directly represses the eve stripe 3+7 enhancer, we found that in the eve stripe 2+7 enhancer, Hunchback repression is prevented by nearby sequences-this phenomenon is called counter-repression. We also found evidence that Caudal binding sites are responsible for counter-repression, and that this interaction may be a conserved feature of eve stripe 2 enhancers. Our results alter the textbook view of eve stripe 2 regulation wherein Hb is described as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted. We discuss how counter-repression may influence eve stripe 2 regulation and evolution.

Quantitative Measurement and Thermodynamic Modeling of Fused Enhancers Support a Two-Tiered Mechanism for Interpreting Regulatory DNA.

Computational models of enhancer function generally assume that transcription factors (TFs) exert their regulatory effects independently, modeling an enhancer as a "bag of sites." These models fail on endogenous loci that harbor multiple enhancers, and a "two-tier" model appears better suited: in each enhancer TFs work independently, and the total expression is a weighted sum of their expression readouts. Here, we test these two opposing views on how cis-regulatory information is integrated. We fused two Drosophila blastoderm enhancers, measured their readouts, and applied the above two models to these data. The two-tier mechanism better fits these readouts, suggesting that these fused enhancers comprise multiple independent modules, despite having sequence characteristics typical of single enhancers. We show that short-range TF-TF interactions are not sufficient to designate such modules, suggesting unknown underlying mechanisms. Our results underscore that mechanisms of how modules are defined and how their outputs are combined remain to be elucidated.

Krüppel Expression Levels Are Maintained through Compensatory Evolution of Shadow Enhancers.

Many developmental genes are controlled by shadow enhancers­pairs of enhancers that drive overlapping expression patterns. We hypothesized that compensatory evolution can maintain the total expression of a gene, while individual shadow enhancers diverge between species. To test this hypothesis, we analyzed expression driven by orthologous pairs of shadow enhancers from Drosophila melanogaster, Drosophila yakuba, and Drosophila pseudoobscura that control expression of Krüppel, a transcription factor that patterns the anterior-posterior axis of blastoderm embryos. We found that the expression driven by the pair of enhancers is conserved between these three species, but expression levels driven by the individual enhancers are not. Using sequence analysis and experimental perturbation, we show that each shadow enhancer is regulated by different transcription factors. These results support the hypothesis that compensatory evolution can occur between shadow enhancers, which has implications for mechanistic and evolutionary studies of gene regulation.

Yearly planning meetings: individualized development plans aren't just more paperwork.

The National Institutes of Health (NIH) encourages trainees to make Individualized Development Plans to help them prepare for academic and nonacademic careers. We describe our approach to building an Individualized Development Plan, the reasons we find them useful and empowering for both PIs and trainees, and resources to help other labs implement them constructively.

Shadow enhancers enable Hunchback bifunctionality in the Drosophila embryo.

Hunchback (Hb) is a bifunctional transcription factor that activates and represses distinct enhancers. Here, we investigate the hypothesis that Hb can activate and repress the same enhancer. Computational models predicted that Hb bifunctionally regulates the even-skipped (eve) stripe 3+7 enhancer (eve3+7) in Drosophila blastoderm embryos. We measured and modeled eve expression at cellular resolution under multiple genetic perturbations and found that the eve3+7 enhancer could not explain endogenous eve stripe 7 behavior. Instead, we found that eve stripe 7 is controlled by two enhancers: the canonical eve3+7 and a sequence encompassing the minimal eve stripe 2 enhancer (eve2+7). Hb bifunctionally regulates eve stripe 7, but it executes these two activities on different pieces of regulatory DNA--it activates the eve2+7 enhancer and represses the eve3+7 enhancer. These two "shadow enhancers" use different regulatory logic to create the same pattern.

A gene expression atlas of a bicoid-depleted Drosophila embryo reveals early canalization of cell fate.

In developing embryos, gene regulatory networks drive cells towards discrete terminal fates, a process called canalization. We studied the behavior of the anterior-posterior segmentation network in Drosophila melanogaster embryos by depleting a key maternal input, bicoid (bcd), and measuring gene expression patterns of the network at cellular resolution. This method results in a gene expression atlas containing the levels of mRNA or protein expression of 13 core patterning genes over six time points for every cell of the blastoderm embryo. This is the first cellular resolution dataset of a genetically perturbed Drosophila embryo that captures all cells in 3D. We describe the technical developments required to build this atlas and how the method can be employed and extended by others. We also analyze this novel dataset to characterize the degree and timing of cell fate canalization in the segmentation network. We find that in two layers of this gene regulatory network, following depletion of bcd, individual cells rapidly canalize towards normal cell fates. This result supports the hypothesis that the segmentation network directly canalizes cell fate, rather than an alternative hypothesis whereby cells are initially mis-specified and later eliminated by apoptosis. Our gene expression atlas provides a high resolution picture of a classic perturbation and will enable further computational modeling of canalization and gene regulation in this transcriptional network.

Comparing mRNA levels using in situ hybridization of a target gene and co-stain.

In situ hybridization is an important technique for measuring the spatial expression patterns of mRNA in cells, tissues, and whole animals. However, mRNA levels cannot be compared across experiments using typical protocols. Here we present a semi-quantitative method to compare mRNA levels of a gene across multiple samples. This method yields an estimate of the error in the measurement to allow statistical comparison. Our method uses a typical in situ hybridization protocol to stain for a target gene and an internal standard, which we refer to as a co-stain. As a proof of concept, we apply this method to multiple lines of transgenic Drosophila embryos, harboring constructs that express reporter genes to different levels. We generated this test set by mutating enhancer sequences to contain different numbers of binding sites for Zelda, a transcriptional activator. We demonstrate that using a co-stain with in situ hybridization is an effective method to compare mRNA levels across samples. This method requires only minor modifications to existing in situ hybridization protocols and uses straightforward analysis techniques. This strategy can be broadly applied to detect quantitative, spatially resolved changes in mRNA levels.

Depleting gene activities in early Drosophila embryos with the "maternal-Gal4-shRNA" system.

In a developing Drosophila melanogaster embryo, mRNAs have a maternal origin, a zygotic origin, or both. During the maternal-zygotic transition, maternal products are degraded and gene expression comes under the control of the zygotic genome. To interrogate the function of mRNAs that are both maternally and zygotically expressed, it is common to examine the embryonic phenotypes derived from female germline mosaics. Recently, the development of RNAi vectors based on short hairpin RNAs (shRNAs) effective during oogenesis has provided an alternative to producing germline clones. Here, we evaluate the efficacies of: (1) maternally loaded shRNAs to knockdown zygotic transcripts and (2) maternally loaded Gal4 protein to drive zygotic shRNA expression. We show that, while Gal4-driven shRNAs in the female germline very effectively generate phenotypes for genes expressed maternally, maternally loaded shRNAs are not very effective at generating phenotypes for early zygotic genes. However, maternally loaded Gal4 protein is very efficient at generating phenotypes for zygotic genes expressed during mid-embryogenesis. We apply this powerful and simple method to unravel the embryonic functions of a number of pleiotropic genes.

Dissecting sources of quantitative gene expression pattern divergence between Drosophila species.

Gene expression patterns can diverge between species due to changes in a gene's regulatory DNA or changes in the proteins, e.g., transcription factors (TFs), that regulate the gene. We developed a modeling framework to uncover the sources of expression differences in blastoderm embryos of three Drosophila species, focusing on the regulatory circuit controlling expression of the hunchback (hb) posterior stripe. Using this framework and cellular-resolution expression measurements of hb and its regulating TFs, we found that changes in the expression patterns of hb's TFs account for much of the expression divergence. We confirmed our predictions using transgenic D. melanogaster lines, which demonstrate that this set of orthologous cis-regulatory elements (CREs) direct similar, but not identical, expression patterns. We related expression pattern differences to sequence changes in the CRE using a calculation of the CRE's TF binding site content. By applying this calculation in both the transgenic and endogenous contexts, we found that changes in binding site content affect sensitivity to regulating TFs and that compensatory evolution may occur in circuit components other than the CRE.

A conserved developmental patterning network produces quantitatively different output in multiple species of Drosophila.

Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3-4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and number of equivalent cells.