Probes for INS

Prevailing dogma holds that cell-cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.

Cardiovascular disease is the leading cause of morbidity and mortality in the Western world owing to the limited regenerative capacity of the mammalian cardiovascular system. In lieu of new muscle synthesis, the human heart replaces necrotic tissue with deposition of a noncontractile scar. By contrast, the adult zebrafish is endowed with a remarkable regenerative capacity, capable of de novo cardiomyocyte (CM) creation and scar tissue removal when challenged with an acute injury. In these studies, we examined the contributions of the dynamically regulated microRNA miR-101a during adult zebrafish heart regeneration. We demonstrate that miR-101a expression is rapidly depleted within 3 days post-amputation (dpa) but is highly upregulated by 7-14 dpa, before returning to uninjured levels at the completion of the regenerative process. Employing heat-inducible transgenic strains and antisense oligonucleotides, we demonstrate that decreases in miR-101a levels at the onset of cardiac injury enhanced CM proliferation. Interestingly, prolonged suppression of miR-101a activity stimulates new muscle synthesis but with defects in scar tissue clearance. Upregulation of miR-101a expression between 7 and 14 dpa is essential to stimulate removal of the scar. Through a series of studies, we identified the proto-oncogene fosab (cfos) as a potent miR-101a target gene, stimulator of CM proliferation, and inhibitor of scar tissue removal. Importantly, combinatorial depletion of fosab and miR-101a activity rescued defects in scar tissue clearance mediated by miR-101a inhibition alone. In summation, our studies indicate that the precise temporal modulation of the miR-101a/fosab genetic axis is crucial for coordinating CM proliferation and scar tissue removal during zebrafish heart regeneration.

Wnt signaling plays an important role in uterine organogenesis and oncogenesis. Our mRNA expression data documents the expression of various Wnt pathway members during the key stages of uterine epithelial gland development. Our data illustrates the expression of Wnt signaling inhibitors (Axin2, Sfrp2, Sfrp4, Dkk1 and Dkk3) in mice uteri at postnatal day 6 (PND 6) and day 15 (PND 15). They also describe the expression pattern of the Wnt ligands (Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt5b, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a and Wnt10b) in mice uteri with or without progesterone treatment. Detailed interpretation and discussion of these data is presented in the research article entitled “Differential Wnt signaling activity limits epithelial gland development to the anti-mesometrial side of the mouse uterus” [1].

Reactive oxygen species (ROS) participate in numerous cell responses, including proliferation, DNA damage, and cell death. Based on these disparate activities, both promotion and inhibition of ROS have been proposed for cancer therapy. However, how the ROS response is determined is not clear. We examined the activities of ROS in a model of Apc deletion, where loss of the Wnt target gene Myc both rescues APC loss and prevents ROS accumulation. Following APC loss, Myc has been shown to up-regulate RAC1 to promote proliferative ROS through NADPH oxidase (NOX). However, APC loss also increased the expression of TIGAR, which functions to limit ROS. To explore this paradox, we used three-dimensional (3D) cultures and in vivo models to show that deletion of TIGAR increased ROS damage and inhibited proliferation. These responses were suppressed by limiting damaging ROS but enhanced by lowering proproliferative NOX-derived ROS. Despite having opposing effects on ROS levels, loss of TIGAR and RAC1 cooperated to suppress intestinal proliferation following APC loss. Our results indicate that the pro- and anti-proliferative effects of ROS can be independently modulated in the same cell, with two key targets in the Wnt pathway functioning to integrate the different ROS signals for optimal cell proliferation.

The chemical and mechanical properties of extracellular matrices (ECMs) modulate diverse aspects of cellular fates; however, how regional heterogeneity in ECM composition regulates developmental programs is not well understood. We discovered that fibronectin 1 (Fn1) is expressed in strikingly non-uniform patterns during mouse development, suggesting that regionalized synthesis of the ECM plays cell-specific regulatory roles during embryogenesis. To test this hypothesis, we ablated Fn1 in the neural crest (NC), a population of multi-potent progenitors expressing high levels of Fn1. We found that Fn1 synthesized by the NC mediated morphogenesis of the aortic arch artery and differentiation of NC cells into vascular smooth muscle cells (VSMCs) by regulating Notch signaling. We show that NC Fn1 signals in an NC cell-autonomous manner through integrin α5β1 expressed by the NC, leading to activation of Notch and differentiation of VSMCs. Our data demonstrate an essential role of the localized synthesis of Fn1 in cardiovascular development and spatial regulation of Notch signaling.

Aging causes cardiac dysfunction, often leading to heart failure and death. The molecular basis of age-associated changes in cardiac structure and function is largely unknown. The fruit fly, Drosophila melanogaster, is well-suited to investigate the genetics of cardiac aging. Flies age rapidly over the course of weeks, benefit from many tools to easily manipulate their genome, and their heart has significant genetic and phenotypic similarities to the human heart. Here, we performed a cardiac-specific gene expression study on aging Drosophila and carried out a comparative meta-analysis with published rodent data. Pathway level transcriptome comparisons suggest that age-related, extra-cellular matrix remodeling and alterations in mitochondrial metabolism, protein handling, and contractile functions are conserved between Drosophila and rodent hearts. However, expression of only a few individual genes similarly changed over time between and even within species. We also examined gene expression in single fly hearts and found significant variability as has been reported in rodents. We propose that individuals may arrive at similar cardiac aging phenotypes via dissimilar transcriptional changes, including those in transcription factors and micro-RNAs. Finally, our data suggest the transcription factor Odd-skipped, which is essential for normal heart development, is also a crucial regulator of cardiac aging.

The epithelial lining of the Fallopian tube is vital for fertility, providing nutrition to gametes, and facilitating their transport. It is composed of two major cell types: secretory cells and ciliated cells. Interestingly, human ovarian cancer precursor lesions are primarily consisting of secretory cells. It is unclear why secretory cells are the dominant cell type in these lesions. Additionally, the underlying mechanisms governing Fallopian tube epithelial homoeostasis are currently unknown. In the present study, we showed that across the different developmental stages of mouse oviduct, secretory cells are the most frequently dividing cells of the oviductal epithelium. In vivo genetic cell lineage tracing showed that secretory cells not only self-renew, but also give rise to ciliated cells. Analysis of a Wnt reporter mouse model and different Wnt target genes showed that the Wnt signaling pathway is involved in oviductal epithelial homoeostasis. By developing two triple transgenic mouse models, we showed that Wnt/β-catenin signaling is essential for self-renewal as well as differentiation of secretory cells. In summary, our results provide mechanistic insight into oviductal epithelial homoeostasis.

The ultimate formation of a four-chambered heart allowing the separation of the pulmonary and systemic circuits was key for the evolutionary success of tetrapods. Complex processes of cell diversification and tissue morphogenesis allow the left and right cardiac compartments to become distinct but remain poorly understood. Here, we describe an unexpected laterality in the single zebrafish atrium analogous to that of the two atria in amniotes, including mammals. This laterality appears to derive from an embryonic antero-posterior asymmetry revealed by the expression of the transcription factor gene meis2b. In adult zebrafish hearts, meis2b expression is restricted to the left side of the atrium where it controls the expression of pitx2c, a regulator of left atrial identity in mammals. Altogether, our studies suggest that the multi-chambered atrium in amniotes arose from a molecular blueprint present before the evolutionary emergence of cardiac septation and provide insights into the establishment of atrial asymmetry.