Multiplexed chromatin imaging reveals predominantly pairwise long-range coordination between Drosophila Polycomb genes.

Polycomb (Pc) group proteins are transcriptional regulators with key roles in development, cell identity, and differentiation. Pc-bound chromatin regions form repressive domains that interact in 3D to assemble repressive nuclear compartments. Here, we use multiplexed chromatin imaging to investigate whether Pc compartments involve the clustering of multiple Pc domains during Drosophila development.

Hi-M: A Multiplex Oligopaint FISH Method to Capture Chromatin Conformations In Situ and Accompanying Open-Source Acquisition Software.

The simultaneous observation of three-dimensional (3D) chromatin structure and transcription in single cells is critical to understand how DNA is organized inside cells and how this organization influences or is affected by other processes, such as transcription. We have recently introduced an innovative technology known as Hi-M, which enables the sequential tagging, 3D visualization, and precise localization of multiple genomic DNA regions alongside RNA expression within individual cells. In this chapter, we present a comprehensive guide outlining the creation of probes, as well as sample preparation and labeling.

pyHiM: a new open-source, multi-platform software package for spatial genomics based on multiplexed DNA-FISH imaging.

Genome-wide ensemble sequencing methods improved our understanding of chromatin organization in eukaryotes but lack the ability to capture single-cell heterogeneity and spatial organization. To overcome these limitations, new imaging-based methods have emerged, giving rise to the field of spatial genomics. Here, we present pyHiM, a user-friendly python toolbox specifically designed for the analysis of multiplexed DNA-FISH data and the reconstruction of chromatin traces in individual cells.

Contributions of 3D chromatin structure to cell-type-specific gene regulation.

Eukaryotic genomes are organized in 3D in a multiscale manner, and different mechanisms acting at each of these scales can contribute to transcriptional regulation. However, the large single-cell variability in 3D chromatin structures represents a challenge to understand how transcription may be differentially regulated between cell types in a robust and efficient manner. Here, we describe the different mechanisms by which 3D chromatin structure was shown to contribute to cell-type-specific transcriptional regulation.

Median eminence blood flow influences food intake by regulating ghrelin access to the metabolic brain.

Central integration of peripheral appetite-regulating signals ensures maintenance of energy homeostasis. Thus, plasticity of circulating molecule access to neuronal circuits involved in feeding behavior plays a key role in the adaptive response to metabolic changes. However, the mechanisms involved remain poorly understood despite their relevance for therapeutic development.

Metabolic Stress Impairs Pericyte Response to Optogenetic Stimulation in Pancreatic Islets.

Pancreatic islets are highly vascularized micro-organs ensuring whole body glucose homeostasis. Islet vascular cells play an integral part in sustaining adequate insulin release by beta cells. In particular, recent studies have demonstrated that islet pericytes regulate local blood flow velocity and are required for maintenance of beta cell maturity and function.

Maternal hypothyroidism in mice influences glucose metabolism in adult offspring.

Aims/hypothesis: During pregnancy, maternal metabolic disease and hormonal imbalance may alter fetal beta cell development and/or proliferation, thus leading to an increased risk for developing type 2 diabetes in adulthood. Although thyroid hormones play an important role in fetal endocrine pancreas development, the impact of maternal hypothyroidism on glucose homeostasis in adult offspring remains poorly understood.

Methods: We investigated this using a mouse model of hypothyroidism, induced by administration of an iodine-deficient diet supplemented with propylthiouracil during gestation.

Integrin β1 Optimizes Diabetogenic T Cell Migration and Function in the Pancreas.

T cell search behavior is dictated by their need to encounter their specific antigen to eliminate target cells. However, mechanisms controlling effector T cell motility are highly tissue-dependent. Specifically, how diabetogenic T cells encounter their target beta cells in dispersed islets throughout the pancreas (PA) during autoimmune diabetes remains unclear.

Imaging endocrinology in animal models of endocrine disease.

Endocrine organs secrete a variety of hormones involved in the regulation of a multitude of body functions. Although pancreatic islets were discovered at the turn of the 19th century, other endocrine glands remained commonly described as diffuse endocrine systems. Over the last two decades, development of new imaging techniques and genetically-modified animals with cell-specific fluorescent tags or specific hormone deficiencies have enabled in vivo imaging of endocrine organs and revealed intricate endocrine cell network structures and plasticity.

CD4 T Helper Cells Play a Key Role in Maintaining Diabetogenic CD8 T Cell Function in the Pancreas.

Autoreactive CD8 and CD4 T cells have been assigned independent key roles in the destruction of insulin-producing beta cells resulting in type 1 diabetes. Although CD4 help for the generation of efficient CD8 T cell responses in lymphoid tissue has been extensively described, whether these two cell populations cooperate in islet destruction remains unclear. By using intravital 2-photon microscopy in a mouse model of diabetes, we visualized both effector T cell populations in the pancreas during disease onset.

Adult muscle-derived stem cells engraft and differentiate into insulin-expressing cells in pancreatic islets of diabetic mice.

Background: Pancreatic beta cells are unique effectors in the control of glucose homeostasis and their deficiency results in impaired insulin production leading to severe diabetic diseases. Here, we investigated the potential of a population of nonadherent muscle-derived stem cells (MDSC) from adult mouse muscle to differentiate in vitro into beta cells when transplanted as undifferentiated stem cells in vivo to compensate for beta-cell deficiency.

Results: In vitro, cultured MDSC spontaneously differentiated into insulin-expressing islet-like cell clusters as revealed using MDSC from transgenic mice expressing GFP or mCherry under the control of an insulin promoter.

Metabolism Regulates Exposure of Pancreatic Islets to Circulating Molecules In Vivo.

Pancreatic β-cells modulate insulin secretion through rapid sensing of blood glucose and integration of gut-derived signals. Increased insulin demand during pregnancy and obesity alters islet function and mass and leads to gestational diabetes mellitus and type 2 diabetes in predisposed individuals. However, it is unclear how blood-borne factors dynamically access the islets of Langerhans.

Palatability Can Drive Feeding Independent of AgRP Neurons.

Feeding behavior is exquisitely regulated by homeostatic and hedonic neural substrates that integrate energy demand as well as the reinforcing and rewarding aspects of food. Understanding the net contribution of homeostatic and reward-driven feeding has become critical because of the ubiquitous source of energy-dense foods and the consequent obesity epidemic. Hypothalamic agouti-related peptide-secreting neurons (AgRP neurons) provide the primary orexigenic drive of homeostatic feeding.

Redistribution of PDGFRβ cells and NG2DsRed pericytes at the cerebrovasculature after status epilepticus.

Purpose: The role of cerebrovascular dysfunction in seizure disorders is recognized. Blood-brain barrier (BBB) damage in epilepsy has been linked to endothelial and glial pathophysiological changes. Little is known about the involvement of pericytes, a cell type that contributes to BBB function.

The blood-brain barrier as a regulator of the gut-brain axis.

The gut-brain axis is involved in metabolic homeostasis through optimization of nutrient absorption and appetite regulation, and encompasses a two-way communication between the gastrointestinal tract and neural circuits in the brain. An important feature of this axis is the secretion of gut-derived peptide hormones which signal energy status to the brain, provoking adaptive behaviors such as food intake or satiation. However, the major integrator of gut signals, the arcuate nucleus of the hypothalamus, is protected by blood-brain barrier, an obstacle to free diffusion of circulating molecules.

Sustained alterations of hypothalamic tanycytes during posttraumatic hypopituitarism in male mice.

Traumatic brain injury is a leading cause of hypopituitarism, which compromises patients' recovery, quality of life, and life span. To date, there are no means other than standardized animal studies to provide insights into the mechanisms of posttraumatic hypopituitarism. We have found that GH levels were impaired after inducing a controlled cortical impact (CCI) in mice.

Sox2(+) stem/progenitor cells in the adult mouse pituitary support organ homeostasis and have tumor-inducing potential.

Sox2(+) adult mouse pituitary cells can self-renew and terminally differentiate in vitro, but their physiological role in vivo and possible contribution to oncogenesis remain largely unknown. Using genetic lineage tracing, we show here that the Sox2(+) cell compartment of both the embryonic and adult pituitary contains stem/progenitor cells that are able to differentiate into all hormone-producing lineages and contribute to organ homeostasis during postnatal life. In addition, we show that targeted expression of oncogenic β-catenin in Sox2(+) cells gives rise to pituitary tumors, but, unexpectedly, the tumor mass is not derived from the Sox2(+) mutation-sustaining cells, suggesting a paracrine role of Sox2(+) cells in pituitary oncogenesis.

Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons.

To maintain homeostasis, hypothalamic neurons in the arcuate nucleus must dynamically sense and integrate a multitude of peripheral signals. Blood-borne molecules must therefore be able to circumvent the tightly sealed vasculature of the blood-brain barrier to rapidly access their target neurons. However, how information encoded by circulating appetite-modifying hormones is conveyed to central hypothalamic neurons remains largely unexplored.

Existence of long-lasting experience-dependent plasticity in endocrine cell networks.

Experience-dependent plasticity of cell and tissue function is critical for survival by allowing organisms to dynamically adjust physiological processes in response to changing or harsh environmental conditions. Despite the conferred evolutionary advantage, it remains unknown whether emergent experience-dependent properties are present in cell populations organized as networks within endocrine tissues involved in regulating body-wide homeostasis. Here we show, using lactation to repeatedly activate a specific endocrine cell network in situ in the mammalian pituitary, that templates of prior demand are permanently stored through stimulus-evoked alterations to the extent and strength of cell-cell connectivity.

Coordination of calcium signals by pituitary endocrine cells in situ.

The pulsatile secretion of hormones from the mammalian pituitary gland drives a wide range of homeostatic responses by dynamically altering the functional set-point of effector tissues. To accomplish this, endocrine cell populations residing within the intact pituitary display large-scale changes in coordinated calcium-spiking activity in response to various hypothalamic and peripheral inputs. Although the pituitary gland is structurally compartmentalized into specific and intermingled endocrine cell networks, providing a clear morphological basis for such coordinated activity, the mechanisms which facilitate the timely propagation of information between cells in situ remain largely unexplored.

Identification of lead compounds targeting the cathepsin B-like enzyme of Eimeria tenella.

Cysteine peptidases have been implicated in the development and pathogenesis of Eimeria. We have identified a single-copy cathepsin B-like cysteine peptidase gene in the genome database of Eimeria tenella (EtCatB). Molecular modeling of the predicted protein suggested that it differs significantly from host enzymes and could be a good drug target.

Influence of estrogens on GH-cell network dynamics in females: a live in situ imaging approach.

The secretion of endocrine hormones from pituitary cells finely regulates a multitude of homeostatic processes. To dynamically adapt to changing physiological status and environmental stimuli, the pituitary gland must undergo marked structural and functional plasticity. Endocrine cell plasticity is thought to primarily rely on variations in cell proliferation and size.

Endocrine cells and blood vessels work in tandem to generate hormone pulses.

Hormones are dynamically collected by fenestrated capillaries to generate pulses, which are then decoded by target tissues to mount a biological response. To generate hormone pulses, endocrine systems have evolved mechanisms to tightly regulate blood perfusion and oxygenation, coordinate endocrine cell responses to secretory stimuli, and regulate hormone uptake from the perivascular space into the bloodstream. Based on recent findings, we review here the mechanisms that exist in endocrine systems to regulate blood flow, and facilitate coordinated cell activity and output under both normal physiological and pathological conditions in the pituitary gland and pancreas.

Functional importance of blood flow dynamics and partial oxygen pressure in the anterior pituitary.

The pulsatile release of hormone is obligatory for the control of a range of important body homeostatic functions. To generate these pulses, endocrine organs have developed finely regulated mechanisms to modulate blood flow both to meet the metabolic demand associated with intense endocrine cell activity and to ensure the temporally precise uptake of secreted hormone into the bloodstream. With a particular focus on the pituitary gland as a model system, we review here the importance of the interplay between blood flow regulation and oxygen tensions in the functioning of endocrine systems, and the known regulatory signals involved in the modification of flow patterns under both normal physiological and pathological conditions.