Engineering synthetic suppressor T cells that execute locally targeted immunoprotective programs.

Immune homeostasis requires a balance of inflammatory and suppressive activities. To design cells potentially useful for local immune suppression, we engineered conventional CD4 T cells with synthetic Notch (synNotch) receptors driving antigen-triggered production of anti-inflammatory payloads. Screening a diverse library of suppression programs, we observed the strongest suppression of cytotoxic T cell attack by the production of both anti-inflammatory factors (interleukin-10, transforming growth factor-β1, programmed death ligand 1) and sinks for proinflammatory cytokines (interleukin-2 receptor subunit CD25).

Single-cell transcriptome and accessible chromatin dynamics during endocrine pancreas development.

Delineating gene regulatory networks that orchestrate cell-type specification is a continuing challenge for developmental biologists. Single-cell analyses offer opportunities to address these challenges and accelerate discovery of rare cell lineage relationships and mechanisms underlying hierarchical lineage decisions. Here, we describe the molecular analysis of mouse pancreatic endocrine cell differentiation using single-cell transcriptomics, chromatin accessibility assays coupled to genetic labeling, and cytometry-based cell purification.

A radial axis defined by semaphorin-to-neuropilin signaling controls pancreatic islet morphogenesis.

The islets of Langerhans are endocrine organs characteristically dispersed throughout the pancreas. During development, endocrine progenitors delaminate, migrate radially and cluster to form islets. Despite the distinctive distribution of islets, spatially localized signals that control islet morphogenesis have not been discovered.

Pathways to clinical CLARITY: volumetric analysis of irregular, soft, and heterogeneous tissues in development and disease.

Three-dimensional tissue-structural relationships are not well captured by typical thin-section histology, posing challenges for the study of tissue physiology and pathology. Moreover, while recent progress has been made with intact methods for clearing, labeling, and imaging whole organs such as the mature brain, these approaches are generally unsuitable for soft, irregular, and heterogeneous tissues that account for the vast majority of clinical samples and biopsies. Here we develop a biphasic hydrogel methodology, which along with automated analysis, provides for high-throughput quantitative volumetric interrogation of spatially-irregular and friable tissue structures.

Research Resource: Genetic Labeling of Human Islet Alpha Cells.

The 2 most abundant human pancreatic islet cell types are insulin-producing β-cells and glucagon-producing α-cells. Defined cis-regulatory elements from rodent Insulin genes have permitted genetic labeling of human islet β-cells, enabling lineage tracing and generation of human β-cell lines, but analogous elements for genetically labeling human α-cells with high specificity do not yet exist. To identify genetic elements that specifically direct reporter expression to human α-cells, we investigated noncoding sequences adjacent to the human GLUCAGON and ARX genes, which are expressed in islet α-cells.

Dissecting Human Gene Functions Regulating Islet Development With Targeted Gene Transduction.

During pancreas development, endocrine precursors and their progeny differentiate, migrate, and cluster to form nascent islets. The transcription factor Neurogenin 3 (Neurog3) is required for islet development in mice, but its role in these dynamic morphogenetic steps has been inferred from fixed tissues. Moreover, little is known about the molecular genetic functions of NEUROG3 in human islet development.

An integrated cell purification and genomics strategy reveals multiple regulators of pancreas development.

The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation.

G2 acquisition by transcription-independent mechanism at the zebrafish midblastula transition.

Following fertilization of many animal embryos, rapid synchronous cleavage divisions give way to longer, asynchronous cell cycles at the midblastula transition (MBT). The cell cycle changes at the MBT, including the addition of gap phases and checkpoint controls, are accompanied by activation of the zygotic genome and the onset of cell motility. Whereas the biochemical changes accompanying the MBT in the vertebrate embryo have been extensively documented, the cellular events are not well understood.

A zebrafish LMO4 ortholog limits the size of the forebrain and eyes through negative regulation of six3b and rx3.

The Six3 and Rx3 homeodomain proteins are essential for the specification and proliferation of forebrain and retinal precursor cells of the vertebrate brain, and the regulatory networks that control their expression are beginning to be elucidated. We identify the zebrafish lmo4b gene as a negative regulator of forebrain growth that acts via restriction of six3 and rx3 expression during early segmentation stages. Loss of lmo4b by morpholino knockdown results in enlargement of the presumptive telencephalon and optic vesicles and an expansion of the post-gastrula expression domains of six3 and rx3.