Shadow Enhancers Are Pervasive Features of Developmental Regulatory Networks

Enhancer redundancy provides phenotypic robustness in mammalian development

Gene enhancer knockout phenotypes and analysis of enhancer activity patterns show that developmental genes are regulated by multiple redundant enhancers in mouse embryos.

Conserved enhancers control notochord expression of vertebrate Brachyury

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

RCCX Genetic Module Theory

NOTE: This page is modified from a summary of findings available at the RCCX and Illness website.

Differences in Krox20-Dependent Regulation of Hoxa2 and Hoxb2 during Hindbrain Development

During hindbrain development, segmental regulation of the paralogous Hoxa2 and Hoxb2 genes in rhombomeres (r) 3 and 5 involves Krox20-dependent enhancers that have been conserved during the duplication of the vertebrate Hox clusters from a common ancestor. Examining these evolutionarily related control regions could provide important insight into the degree to which the basic Krox20-dependent mechanisms, cis-regulatory components, and their organization have been conserved. Toward this goal we have performed a detailed functional analysis of a mouse Hoxa2 enhancer capable of directing reporter expression in r3 and r5. The combined activities of five separate cis-regions, in addition to the conserved Krox20 binding sites, are involved in mediating enhancer function. A CTTT (BoxA) motif adjacent to the Krox20 binding sites is important for r3/r5 activity. The BoxA motif is similar to one (Box1) found in the Hoxb2 enhancer and indicates that the close proximity of these Box motifs to Krox20 sites is a common feature of Krox20 targets in vivo. Two other rhombomeric elements (RE1 and RE3) are essential for r3/r5 activity and share common TCT motifs, indicating that they interact with a similar cofactor(s). TCT motifs are also found in the Hoxb2 enhancer, suggesting that they may be another common feature of Krox20-dependent control regions. The two remaining Hoxa2 cis-elements, RE2 and RE4, are not conserved in the Hoxb2 enhancer and define differences in some of components that can contribute to the Krox20-dependent activities of these enhancers. Furthermore, analysis of regulatory activities of these enhancers in a Krox20 mutant background has uncovered differences in their degree of dependence upon Krox20 for segmental expression. Together, this work has revealed a surprising degree of complexity in the number of cis-elements and regulatory components that contribute to segmental expression mediated by Krox20 and sheds light on the diversity and evolution of Krox20 target sites and Hox regulatory elements in vertebrates.

Targeted mutagenesis of specific genomic DNA sequences in animals for the in vivo generation of variant libraries

Understanding how the number, placement and affinity of transcription factor binding sites dictates gene regulatory programs remains a major unsolved challenge in biology, particularly in the context of multicellular organisms. To uncover these rules, it is first necessary to find the binding sites within a regulatory region with high precision, and then to systematically modulate this binding site arrangement while simultaneously measuring the effect of this modulation on output gene expression. Massively parallel reporter assays (MPRAs), where the gene expression stemming from 10,000s of in vitro-generated regulatory sequences is measured, have made this feat possible in high-throughput in single cells in culture. However, because of lack of technologies to incorporate DNA libraries, MPRAs are limited in whole organisms. To enable MPRAs in multicellular organisms, we generated tools to create a high degree of mutagenesis in specific genomic loci in vivo using base editing. Targeting GFP integrated in the genome of Drosophila cell culture and whole animals as a case study, we show that the base editor AIDevoCDA1 stemming from sea lamprey fused to nCas9 is highly mutagenic. Surprisingly, longer gRNAs increase mutation efficiency and expand the mutating window, which can allow the introduction of mutations in previously untargetable sequences. Finally, we demonstrate arrays of >20 gRNAs that can efficiently introduce mutations along a 200bp sequence, making it a promising tool to test enhancer function in vivo in a high throughput manner.

Evolution as fitness landscape navigation: Concepts, Measures, and...

Fitness landscapes are mappings between genotypes, phenotypes, and fitness that shape evolution. In recent years, empirical work and theoretical models have greatly advanced our understanding of...

Zebrafish hox paralogue group 2 genes function redundantly as selector genes to pattern the second pharyngeal arch

The pharyngeal arches are one of the defining features of the vertebrates, with the first arch forming the mandibles of the jaw and the second forming jaw support structures. The cartilaginous elements of each arch are formed from separate migratory neural crest cell streams, which derive from the dorsal aspect of the neural tube. The second and more posterior crest streams are characterized by specific Hox gene expression. The zebrafish has a larger overall number of Hox genes than the tetrapod vertebrates, as the result of a duplication event in its lineage. However, in both zebrafish and mouse, there are just two members of Hox paralogue group 2 (PG2): Hoxa2 and Hoxb2. Here, we show that morpholino-mediated "knock-down" of both zebrafish Hox PG2 genes results in major defects in second pharyngeal arch cartilages, involving replacement of ventral elements with a mirror-image duplication of first arch structures, and accompanying changes to pharyngeal musculature. In the mouse, null mutants of Hoxa2 have revealed that this single Hox gene is required for normal second arch patterning. By contrast, loss-of-function of either zebrafish Hox PG2 gene individually has no phenotypic consequence, showing that these two genes function redundantly to confer proper pattern to the second pharyngeal arch. We have also used hoxb1a mis-expression to induce localized ectopic expression of zebrafish Hox PG2 genes in the first arch; using this strategy, we find that ectopic expression of either Hox PG2 gene can confer second arch identity onto first arch structures, suggesting that the zebrafish Hox PG2 genes act as "selector genes."

Zebrafish Hox Paralogue Group 2 Genes Function Redundantly as Selector Genes to Pattern the Second Pharyngeal Arch

The pharyngeal arches are one of the defining features of the vertebrates, with the first arch forming the mandibles of the jaw and the second forming jaw support structures. The cartilaginous elements of each arch are formed from separate migratory neural crest cell streams, which derive from the dorsal aspect of the neural tube. The second and more posterior crest streams are characterized by specific Hox gene expression. The zebrafish has a larger overall number of Hox genes than the tetrapod vertebrates, as the result of a duplication event in its lineage. However, in both zebrafish and mouse, there are just two members of Hox paralogue group 2 (PG2): Hoxa2 and Hoxb2. Here, we show that morpholino-mediated “knock-down” of both zebrafish Hox PG2 genes results in major defects in second pharyngeal arch cartilages, involving replacement of ventral elements with a mirror-image duplication of first arch structures, and accompanying changes to pharyngeal musculature. In the mouse, null mutants of Hoxa2 have revealed that this single Hox gene is required for normal second arch patterning. By contrast, loss-of-function of either zebrafish Hox PG2 gene individually has no phenotypic consequence, showing that these two genes function redundantly to confer proper pattern to the second pharyngeal arch. We have also used hoxb1a mis-expression to induce localized ectopic expression of zebrafish Hox PG2 genes in the first arch; using this strategy, we find that ectopic expression of either Hox PG2 gene can confer second arch identity onto first arch structures, suggesting that the zebrafish Hox PG2 genes act as “selector genes.”

Prime editing-installed suppressor tRNAs for disease-agnostic genome editing

Precise genome-editing technologies such as base editing1,2 and prime editing3 can correct most pathogenic gene variants, but their widespread clinical application is impeded by the need to develop new therapeutic agents for each mutation. For diseases that are caused by premature stop codons, suppressor tRNAs (sup-tRNAs) offer a more general strategy. Existing approaches to use sup-tRNAs therapeutically, however, require lifelong administration4,5 or show modest potency, necessitating potentially toxic overexpression. Here we present prime editing-mediated readthrough of premature termination codons (PERT), a strategy to rescue nonsense mutations in a disease-agnostic manner by using prime editing to permanently convert a dispensable endogenous tRNA into an optimized sup-tRNA. Iterative screening of thousands of variants of all 418 human tRNAs identified tRNAs with the strongest sup-tRNA potential. We optimized prime editing agents to install an engineered sup-tRNA at a single genomic locus without overexpression and observed efficient readthrough of premature termination codons and protein rescue in human cell models of Batten disease, Tay-Sachs disease and cystic fibrosis. In vivo delivery of a single prime editor that converts an endogenous mouse tRNA into a sup-tRNA extensively rescued disease pathology in a model of Hurler syndrome. PERT did not induce detected readthrough of natural stop codons or cause significant transcriptomic or proteomic changes. Our findings suggest the potential of disease-agnostic therapeutic genome-editing approaches that require only a single composition of matter to treat diverse genetic diseases.

Box Shadows - Generate CSS Box Shadows

A CSS box-shadow library and generator to create, test and share box shadows.

Gene regulatory networks: from correlative models to causal explanations

Gene regulatory networks (GRNs) explain how the genome controls cellular behaviour and tissue morphogenesis, serving to connect molecular mechanism to functional output. Single-cell technologies now provide descriptions of these networks with unprecedented detail, but this advance has also revealed gene regulatory systems that are too complex for our existing conceptual frameworks. GRNs, which should provide mechanistic explanations, are increasingly reduced to statistical correlations — ‘hairballs’ that fail to capture molecular causation. Here, we explore why this dilemma exists and propose a path forward. We argue that methods in ‘representation learning’ can be used to model GRNs, without needing to capture every molecular detail. For this framework, we advocate three linked principles: models must be inherently mechanistic, with structures grounded in cellular and evolutionary biology; molecular principles and constraints must be used to reduce the solution space for learning GRN models; and more sophisticated forms of experimental perturbation and synthetic biological engineering are needed to train models and test predictions. By reimagining GRNs through these principles, we can bridge the gap from data abundance to new conceptual understanding.

(PDF) Highly efficient genome editing using oocyte-specific z cas9 transgenic zebrafish

PDF | Since its first application, CRISPR/Cas9 rapidly becomes a routine technique to perform genome editing in a variety of biological systems. To... | Find, read and cite all the research you need on ResearchGate

Embryo-scale reverse genetics at single-cell resolution

The maturation of single-cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos1–4. A majority of these data, however, has been collected from wild-type embryos without an appreciation for the latent variation that is present in development. Here we present the ‘zebrafish single-cell atlas of perturbed embryos’: single-cell transcriptomic data from 1,812 individually resolved developing zebrafish embryos, encompassing 19 timepoints, 23 genetic perturbations and a total of 3.2 million cells. The high degree of replication in our study (eight or more embryos per condition) enables us to estimate the variance in cell type abundance organism-wide and to detect perturbation-dependent deviance in cell type composition relative to wild-type embryos. Our approach is sensitive to rare cell types, resolving developmental trajectories and genetic dependencies in the cranial ganglia neurons, a cell population that comprises less than 1% of the embryo. Additionally, time-series profiling of individual mutants identified a group of brachyury-independent cells with strikingly similar transcriptomes to notochord sheath cells, leading to new hypotheses about early origins of the skull. We anticipate that standardized collection of high-resolution, organism-scale single-cell data from large numbers of individual embryos will enable mapping of the genetic dependencies of zebrafish cell types, while also addressing longstanding challenges in developmental genetics, including the cellular and transcriptional plasticity underlying phenotypic diversity across individuals.

A unified network systems approach uncovers a core program underlying T follicular helper cell differentiation

Characterizing multi-scale processes underlying immune-state progression is critical for defining their function. This becomes pertinent for functionally diverse and plastic immune cells, such as T follicular helper (Tfh) cells. Here, we adopt a multi-scale network-systems approach that incorporates both regulatory and protein-protein interactions. This approach integrates diverse data types, captures regulation across levels of immune system organization, and recapitulates known Tfh differentiation drivers. Further, we present CoreNet, a core Tfh gene set that is conserved between humans and mice, across tissue types and disease contexts, and is consistent across data modalities. Using CoreNet, we implicate NR3C1 and interleukin (IL)-12 in the regulation of Tfh differentiation. Notably, IL-12 is permissive for differentiation of Tfh precursors but blocks differentiation into germinal center Tfh cells. Overall, this work elucidates networks with unexplored roles governing Tfh differentiation across species and tissues, while providing a generalizable framework. CoreNet is accessible through an interactive web server: https://pitt-csi.shinyapps.io/tfhcorenet/.

Highly Efficient CRISPR-Cas9-Based Methods for Generating Deletion Mutations and F0 Embryos that Lack Gene Function in Zebrafish

Cas9 RNP complexes consisting of synthetic crRNA:tracrRNA duplex guide RNAs consistently induce mutations in virtually all copies of a targeted gene in zebrafish embryos. Hoshijima et al. show these tools allow effective screening of individual or combinations of gene function in F0 embryos and the facile induction of deletion mutations.

Shadowing

Shadowing is a qualitative user research method in which a researcher observes participants in real‑life contexts over an extended period without interfering. It is used to gain deep contextual insights into behaviors, workflows, environments, emotions, and workarounds.