Spatiotemporal immune atlas of a clinical-grade gene-edited pig-to-human kidney xenotransplant.

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. Here, we transplant a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and study the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells are uncommon in the porcine kidney cortex early after xenotransplantation and consist of primarily myeloid cells.

Inhibition of NFAT promotes loss of tissue resident uterine natural killer cells and attendant pregnancy complications in humans.

Unlabelled: Uterine natural killer cells (uNKs) are a tissue resident lymphocyte population that are critical for pregnancy success. Although mouse models have demonstrated that NK deficiency results in abnormal placentation and poor pregnancy outcomes, the generalizability of this knowledge to humans remains unclear. Here we identify uterus transplant (UTx) recipients as a human population with reduced uNK cells and altered pregnancy phenotypes.

Spatiotemporal immune atlas of the first clinical-grade, gene-edited pig-to-human kidney xenotransplant.

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. We transplanted a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and studied the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells were uncommon in the porcine kidney cortex early after xenotransplantation and consisted of primarily myeloid cells.

Using Single Molecule RNA FISH to Determine Nuclear Export and Transcription Phenotypes in Drosophila Tissues.

Single molecule RNA fluorescence in situ hybridization (smRNA FISH) is a widely used method for examining cellular localization of RNA and assessing gene expression outputs. The Nuclear Pore Complex (NPC) is a nuclear macro-complex known to both mediate nucleocytoplasmic transport and influence transcription via interactions with chromatin. Consequently, depletion of NPC proteins can result in defects in either transcription or nuclear export of mRNA.

Nup98-dependent transcriptional memory is established independently of transcription.

Cellular ability to mount an enhanced transcriptional response upon repeated exposure to external cues is termed transcriptional memory, which can be maintained epigenetically through cell divisions and can depend on a nuclear pore component Nup98. The majority of mechanistic knowledge on transcriptional memory has been derived from bulk molecular assays. To gain additional perspective on the mechanism and contribution of Nup98 to memory, we used single-molecule RNA FISH (smFISH) to examine the dynamics of transcription in cells upon repeated exposure to the steroid hormone ecdysone.

Correct dosage of X chromosome transcription is controlled by a nuclear pore component.

Dosage compensation in Drosophila melanogaster involves a 2-fold transcriptional upregulation of the male X chromosome, which relies on the X-chromosome-binding males-specific lethal (MSL) complex. However, how such 2-fold precision is accomplished remains unclear. Here, we show that a nuclear pore component, Mtor, is involved in setting the correct levels of transcription from the male X chromosome.

BMP heterodimers signal via distinct type I receptor class functions.

Heterodimeric TGF-β ligands outperform homodimers in a variety of developmental, cell culture, and therapeutic contexts; however, the mechanisms underlying this increased potency remain uncharacterized. Here, we use dorsal-ventral axial patterning of the zebrafish embryo to interrogate the BMP2/7 heterodimer signaling mechanism. We demonstrate that differential interactions with BMP antagonists do not account for the reduced signaling ability of homodimers.

p53 mediates target gene association with nuclear speckles for amplified RNA expression.

Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. Although links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor.

Preparation of Polytene Chromosomes, Followed by Immunofluorescence Analysis of Chromatin Structure by Multi-fluorescence Correlations.

larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal reproducibility and low sample success rate as accepted caveats. However, low sample success rates are an obvious disadvantage when polytene chromosomes are used for more high-throughput approaches, such as genetic or antibody screens, or for experiments requiring high-quality chromosome structure preservation.

Genetic Variation in Type 1 Diabetes Reconfigures the 3D Chromatin Organization of T Cells and Alters Gene Expression.

Genetics is a major determinant of susceptibility to autoimmune disorders. Here, we examined whether genome organization provides resilience or susceptibility to sequence variations, and how this would contribute to the molecular etiology of an autoimmune disease. We generated high-resolution maps of linear and 3D genome organization in thymocytes of NOD mice, a model of type 1 diabetes (T1D), and the diabetes-resistant C57BL/6 mice.

Chromatin targeting of nuclear pore proteins induces chromatin decondensation.

Nuclear pore complexes have emerged in recent years as chromatin-binding nuclear scaffolds, able to influence target gene expression. However, how nucleoporins (Nups) exert this control remains poorly understood. Here we show that ectopically tethering Nups, especially Sec13, to chromatin is sufficient to induce chromatin decondensation.

Spatiotemporal Patterning of Zygotic Genome Activation in a Model Vertebrate Embryo.

A defining feature of early embryogenesis is the transition from maternal to zygotic control. This transition requires embryo-wide zygotic genome activation (ZGA), but the extent of spatiotemporal coordination of ZGA between individual cells is unknown. Multiple interrelated parameters, including elapsed time, completed cycles of cell division, and cell size may impact ZGA onset; however, the principal determinant of ZGA during vertebrate embryogenesis is debated.

Oncogenic Notch Promotes Long-Range Regulatory Interactions within Hyperconnected 3D Cliques.

Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning.

Diverse Spatial Expression Patterns Emerge from Unified Kinetics of Transcriptional Bursting.

How transcriptional bursting relates to gene regulation is a central question that has persisted for more than a decade. Here, we measure nascent transcriptional activity in early Drosophila embryos and characterize the variability in absolute activity levels across expression boundaries. We demonstrate that boundary formation follows a common transcription principle: a single control parameter determines the distribution of transcriptional activity, regardless of gene identity, boundary position, or enhancer-promoter architecture.

Single mRNA Molecule Detection in Drosophila.

Single molecule fluorescent in situ hybridization (smFISH) enables quantitative measurements of gene expression and mRNA localization. The technique is increasingly popular for analysis of cultured cells but is not widely applied to intact organisms. Here, we describe a method for labeling and detection of single mRNA molecules in whole embryos of the fruit fly Drosophila melanogaster.

Only accessible information is useful: insights from gradient-mediated patterning.

Information theory is gaining popularity as a tool to characterize performance of biological systems. However, information is commonly quantified without reference to whether or how a system could extract and use it; as a result, information-theoretic quantities are easily misinterpreted. Here, we take the example of pattern-forming developmental systems which are commonly structured as cascades of sequential gene expression steps.

Independent and coordinate trafficking of single Drosophila germ plasm mRNAs.

Messenger RNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. Whereas different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single-molecule imaging, we analysed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells.

The embryo as a laboratory: quantifying transcription in Drosophila.

Transcriptional regulation of gene expression is fundamental to most cellular processes, including determination of cellular fates. Quantitative studies of transcription in cultured cells have led to significant advances in identifying mechanisms underlying transcriptional control. Recent progress allowed implementation of these same quantitative methods in multicellular organisms to ask how transcriptional regulation unfolds both in vivo and at the single molecule level in the context of embryonic development.

Maternal origins of developmental reproducibility.

Cell fate decisions during multicellular development are precisely coordinated, leading to highly reproducible macroscopic structural outcomes [1-3]. The origins of this reproducibility are found at the molecular level during the earliest stages of development when patterns of morphogen molecules emerge reproducibly [4, 5]. However, although the initial conditions for these early stages are determined by the female during oogenesis, it is unknown whether reproducibility is perpetuated from oogenesis or reacquired by the zygote.

Precise developmental gene expression arises from globally stochastic transcriptional activity.

Early embryonic patterning events are strikingly precise, a fact that appears incompatible with the stochastic gene expression observed across phyla. Using single-molecule mRNA quantification in Drosophila embryos, we determine the magnitude of fluctuations in the expression of four critical patterning genes. The accumulation of mRNAs is identical across genes and fluctuates by only ∼8% between neighboring nuclei, generating precise protein distributions.

Sorting sloppy Sonic.

In the classic picture of morphogen-mediated patterning, cells acquire the correct spatial arrangement of specified fates by reading a precisely distributed gradient of morphogen. Xiong et al. now provide evidence for an alternate strategy-cells of the zebrafish neural tube actively sort to their correct positions following disordered specification by Sonic hedgehog.

Shifting patterns: merging molecules, morphogens, motility, and methodology.

We highlight crucial technological progress of the past ten years that permits quantitative analysis of cellular behavior. Adapting these methods to the study of embryogenesis will be essential to advance our understanding of development in the coming decade.

The formation of the Bicoid morphogen gradient requires protein movement from anteriorly localized mRNA.

The Bicoid morphogen gradient directs the patterning of cell fates along the anterior-posterior axis of the syncytial Drosophila embryo and serves as a paradigm of morphogen-mediated patterning. The simplest models of gradient formation rely on constant protein synthesis and diffusion from anteriorly localized source mRNA, coupled with uniform protein degradation. However, currently such models cannot account for all known gradient characteristics.

The fibrodysplasia ossificans progressiva R206H ACVR1 mutation activates BMP-independent chondrogenesis and zebrafish embryo ventralization.

Patients with classic fibrodysplasia ossificans progressiva, a disorder characterized by extensive extraskeletal endochondral bone formation, share a recurrent mutation (R206H) within the glycine/serine-rich domain of ACVR1/ALK2, a bone morphogenetic protein type I receptor. Through a series of in vitro assays using several mammalian cell lines and chick limb bud micromass cultures, we determined that mutant R206H ACVR1 activated BMP signaling in the absence of BMP ligand and mediated BMP-independent chondrogenesis that was enhanced by BMP. We further investigated the interaction of mutant R206H ACVR1 with FKBP1A, a glycine/serine domain-binding protein that prevents leaky BMP type I receptor activation in the absence of ligand.