Publisher Correction: Guided self-organization and cortical plate formation in human brain organoids.

This corrects the article DOI: 10.1038/nbt.3906.

Uncoupling evolutionary changes in DNA sequence, transcription factor occupancy and enhancer activity.

Sequence variation within enhancers plays a major role in both evolution and disease, yet its functional impact on transcription factor (TF) occupancy and enhancer activity remains poorly understood. Here, we assayed the binding of five essential TFs over multiple stages of embryogenesis in two distant Drosophila species (with 1.4 substitutions per neutral site), identifying thousands of orthologous enhancers with conserved or diverged combinatorial occupancy. We used these binding signatures to dissect two properties of developmental enhancers: (1) potential TF cooperativity, using signatures of co-associations and co-divergence in TF occupancy. This revealed conserved combinatorial binding despite sequence divergence, suggesting protein-protein interactions sustain conserved collective occupancy. (2) Enhancer in-vivo activity, revealing orthologous enhancers with conserved activity despite divergence in TF occupancy. Taken together, we identify enhancers with diverged motifs yet conserved occupancy and others with diverged occupancy yet conserved activity, emphasising the need to functionally measure the effect of divergence on enhancer activity.

Guided self-organization and cortical plate formation in human brain organoids.

Three-dimensional cell culture models have either relied on the self-organizing properties of mammalian cells or used bioengineered constructs to arrange cells in an organ-like configuration. While self-organizing organoids excel at recapitulating early developmental events, bioengineered constructs reproducibly generate desired tissue architectures. Here, we combine these two approaches to reproducibly generate human forebrain tissue while maintaining its self-organizing capacity. We use poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments as a floating scaffold to generate elongated embryoid bodies. Microfilament-engineered cerebral organoids (enCORs) display enhanced neuroectoderm formation and improved cortical development. Furthermore, reconstitution of the basement membrane leads to characteristic cortical tissue architecture, including formation of a polarized cortical plate and radial units. Thus, enCORs model the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration. Our data demonstrate that combining 3D cell culture with bioengineering can increase reproducibility and improve tissue architecture.

Qualitative Dynamical Modelling Can Formally Explain Mesoderm Specification and Predict Novel Developmental Phenotypes.

Given the complexity of developmental networks, it is often difficult to predict the effect of genetic perturbations, even within coding genes. Regulatory factors generally have pleiotropic effects, exhibit partially redundant roles, and regulate highly interconnected pathways with ample cross-talk. Here, we delineate a logical model encompassing 48 components and 82 regulatory interactions involved in mesoderm specification during Drosophila development, thereby providing a formal integration of all available genetic information from the literature. The four main tissues derived from mesoderm correspond to alternative stable states. We demonstrate that the model can predict known mutant phenotypes and use it to systematically predict the effects of over 300 new, often non-intuitive, loss- and gain-of-function mutations, and combinations thereof. We further validated several novel predictions experimentally, thereby demonstrating the robustness of model. Logical modelling can thus contribute to formally explain and predict regulatory outcomes underlying cell fate decisions.

Shadow Enhancers Are Pervasive Features of Developmental Regulatory Networks.

Embryogenesis is remarkably robust to segregating mutations and environmental variation; under a range of conditions, embryos of a given species develop into stereotypically patterned organisms. Such robustness is thought to be conferred, in part, through elements within regulatory networks that perform similar, redundant tasks. Redundant enhancers (or "shadow" enhancers), for example, can confer precision and robustness to gene expression, at least at individual, well-studied loci. However, the extent to which enhancer redundancy exists and can thereby have a major impact on developmental robustness remains unknown. Here, we systematically assessed this, identifying over 1,000 predicted shadow enhancers during Drosophila mesoderm development. The activity of 23 elements, associated with five genes, was examined in transgenic embryos, while natural structural variation among individuals was used to assess their ability to buffer against genetic variation. Our results reveal three clear properties of enhancer redundancy within developmental systems. First, it is much more pervasive than previously anticipated, with 64% of loci examined having shadow enhancers. Their spatial redundancy is often partial in nature, while the non-overlapping function may explain why these enhancers are maintained within a population. Second, over 70% of loci do not follow the simple situation of having only two shadow enhancers-often there are three (rols), four (CadN and ade5), or five (Traf1), at least one of which can be deleted with no obvious phenotypic effects. Third, although shadow enhancers can buffer variation, patterns of segregating variation suggest that they play a more complex role in development than generally considered.

A transcription factor collective defines cardiac cell fate and reflects lineage history.

Cell fate decisions are driven through the integration of inductive signals and tissue-specific transcription factors (TFs), although the details on how this information converges in cis remain unclear. Here, we demonstrate that the five genetic components essential for cardiac specification in Drosophila, including the effectors of Wg and Dpp signaling, act as a collective unit to cooperatively regulate heart enhancer activity, both in vivo and in vitro. Their combinatorial binding does not require any specific motif orientation or spacing, suggesting an alternative mode of enhancer function whereby cooperative activity occurs with extensive motif flexibility. A fraction of enhancers co-occupied by cardiogenic TFs had unexpected activity in the neighboring visceral mesoderm but could be rendered active in heart through single-site mutations. Given that cardiac and visceral cells are both derived from the dorsal mesoderm, this "dormant" TF binding signature may represent a molecular footprint of these cells' developmental lineage.

Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development.

Chromatin modifications are associated with many aspects of gene expression, yet their role in cellular transitions during development remains elusive. Here, we use a new approach to obtain cell type-specific information on chromatin state and RNA polymerase II (Pol II) occupancy within the multicellular Drosophila melanogaster embryo. We directly assessed the relationship between chromatin modifications and the spatio-temporal activity of enhancers. Rather than having a unique chromatin state, active developmental enhancers show heterogeneous histone modifications and Pol II occupancy. Despite this complexity, combined chromatin signatures and Pol II presence are sufficient to predict enhancer activity de novo. Pol II recruitment is highly predictive of the timing of enhancer activity and seems dependent on the timing and location of transcription factor binding. Chromatin modifications typically demarcate large regulatory regions encompassing multiple enhancers, whereas local changes in nucleosome positioning and Pol II occupancy delineate single active enhancers. This cell type-specific view identifies dynamic enhancer usage, an essential step in deciphering developmental networks.

Combinatorial binding leads to diverse regulatory responses: Lmd is a tissue-specific modulator of Mef2 activity.

Understanding how complex patterns of temporal and spatial expression are regulated is central to deciphering genetic programs that drive development. Gene expression is initiated through the action of transcription factors and their cofactors converging on enhancer elements leading to a defined activity. Specific constellations of combinatorial occupancy are therefore often conceptualized as rigid binding codes that give rise to a common output of spatio-temporal expression. Here, we assessed this assumption using the regulatory input of two essential transcription factors within the Drosophila myogenic network. Mutations in either Myocyte enhancing factor 2 (Mef2) or the zinc-finger transcription factor lame duck (lmd) lead to very similar defects in myoblast fusion, yet the underlying molecular mechanism for this shared phenotype is not understood. Using a combination of ChIP-on-chip analysis and expression profiling of loss-of-function mutants, we obtained a global view of the regulatory input of both factors during development. The majority of Lmd-bound enhancers are co-bound by Mef2, representing a subset of Mef2's transcriptional input during these stages of development. Systematic analyses of the regulatory contribution of both factors demonstrate diverse regulatory roles, despite their co-occupancy of shared enhancer elements. These results indicate that Lmd is a tissue-specific modulator of Mef2 activity, acting as both a transcriptional activator and repressor, which has important implications for myogenesis. More generally, this study demonstrates considerable flexibility in the regulatory output of two factors, leading to additive, cooperative, and repressive modes of co-regulation.

Model-based method for transcription factor target identification with limited data.

We present a computational method for identifying potential targets of a transcription factor (TF) using wild-type gene expression time series data. For each putative target gene we fit a simple differential equation model of transcriptional regulation, and the model likelihood serves as a score to rank targets. The expression profile of the TF is modeled as a sample from a Gaussian process prior distribution that is integrated out using a nonparametric Bayesian procedure. This results in a parsimonious model with relatively few parameters that can be applied to short time series datasets without noticeable overfitting. We assess our method using genome-wide chromatin immunoprecipitation (ChIP-chip) and loss-of-function mutant expression data for two TFs, Twist, and Mef2, controlling mesoderm development in Drosophila. Lists of top-ranked genes identified by our method are significantly enriched for genes close to bound regions identified in the ChIP-chip data and for genes that are differentially expressed in loss-of-function mutants. Targets of Twist display diverse expression profiles, and in this case a model-based approach performs significantly better than scoring based on correlation with TF expression. Our approach is found to be comparable or superior to ranking based on mutant differential expression scores. Also, we show how integrating complementary wild-type spatial expression data can further improve target ranking performance.

Temporal ChIP-on-chip reveals Biniou as a universal regulator of the visceral muscle transcriptional network.

Smooth muscle plays a prominent role in many fundamental processes and diseases, yet our understanding of the transcriptional network regulating its development is very limited. The FoxF transcription factors are essential for visceral smooth muscle development in diverse species, although their direct regulatory role remains elusive. We present a transcriptional map of Biniou (a FoxF transcription factor) and Bagpipe (an Nkx factor) activity, as a first step to deciphering the developmental program regulating Drosophila visceral muscle development. A time course of chromatin immunoprecipitatation followed by microarray analysis (ChIP-on-chip) experiments and expression profiling of mutant embryos reveal a dynamic map of in vivo bound enhancers and direct target genes. While Biniou is broadly expressed, it regulates enhancers driving temporally and spatially restricted expression. In vivo reporter assays indicate that the timing of Biniou binding is a key trigger for the time span of enhancer activity. Although bagpipe and biniou mutants phenocopy each other, their regulatory potential is quite different. This network architecture was not apparent from genetic studies, and highlights Biniou as a universal regulator in all visceral muscle, regardless of its developmental origin or subsequent function. The regulatory connection of a number of Biniou target genes is conserved in mice, suggesting an ancient wiring of this developmental program.