Coronary Revascularization During Heart Regeneration Is Regulated by Epicardial and Endocardial Cues and Forms a Scaffold for Cardiomyocyte Repopulation.

Defective coronary network function and insufficient blood supply are both cause and consequence of myocardial infarction. Efficient revascularization after infarction is essential to support tissue repair and function. Zebrafish hearts exhibit a remarkable ability to regenerate, and coronary revascularization initiates within hours of injury, but how this process is regulated remains unknown. Here, we show that revascularization requires a coordinated multi-tissue response culminating with the formation of a complex vascular network available as a scaffold for cardiomyocyte repopulation. During a process we term "coronary-endocardial anchoring," new coronaries respond by sprouting (1) superficially within the regenerating epicardium and (2) intra-ventricularly toward the activated endocardium. Mechanistically, superficial revascularization is guided by epicardial Cxcl12-Cxcr4 signaling and intra-ventricular sprouting by endocardial Vegfa signaling. Our findings indicate that the injury-activated epicardium and endocardium support cardiomyocyte replenishment initially through the guidance of coronary sprouting. Simulating this process in the injured mammalian heart should help its healing.

Visualization of Neuregulin 1 ectodomain shedding reveals its local processing in vitro and in vivo.

Neuregulin1 (NRG1) plays diverse developmental roles and is likely involved in several neurological disorders including schizophrenia. The transmembrane NRG1 protein is proteolytically cleaved and released as a soluble ligand for ErbB receptors. Such post-translational processing, referred to as 'ectodomain shedding', is thought to be crucial for NRG1 function. However, little is known regarding the regulatory mechanism of NRG1 cleavage in vivo. Here, we developed a fluorescent probe, NRG1 Cleavage Indicating SenSOR (N-CISSOR), by fusing mCherry and GFP to the extracellular and intracellular domains of NRG1, respectively. N-CISSOR mimicked the subcellular localization and biochemical properties of NRG1 including cleavage dynamics and ErbB phosphorylation in cultured cells. mCherry/GFP ratio imaging of phorbol-12-myristate-13-acetate-stimulated N-CISSOR-expressing HEK293T cells enabled to monitor rapid ectodomain shedding of NRG1 at the subcellular level. Utilizing N-CISSOR in zebrafish embryos revealed preferential axonal NRG1 ectodomain shedding in developing motor neurons, demonstrating that NRG1 ectodomain shedding is spatially regulated at the subcellular level. Thus, N-CISSOR will be a valuable tool for elucidating the spatiotemporal regulation of NRG1 ectodomain shedding, both in vitro and in vivo.

Neuregulin 1 Type II-ErbB Signaling Promotes Cell Divisions Generating Neurons from Neural Progenitor Cells in the Developing Zebrafish Brain.

Post-mitotic neurons are generated from neural progenitor cells (NPCs) at the expense of their proliferation. Molecular and cellular mechanisms that regulate neuron production temporally and spatially should impact on the size and shape of the brain. While transcription factors such as neurogenin1 (neurog1) and neurod govern progression of neurogenesis as cell-intrinsic mechanisms, recent studies show regulatory roles of several cell-extrinsic or intercellular signaling molecules including Notch, FGF and Wnt in production of neurons/neural progenitor cells from neural stem cells/radial glial cells (NSCs/RGCs) in the ventricular zone (VZ). However, it remains elusive how production of post-mitotic neurons from neural progenitor cells is regulated in the sub-ventricular zone (SVZ). Here we show that newborn neurons accumulate in the basal-to-apical direction in the optic tectum (OT) of zebrafish embryos. While neural progenitor cells are amplified by mitoses in the apical ventricular zone, neurons are exclusively produced through mitoses of neural progenitor cells in the sub-basal zone, later in the sub-ventricular zone, and accumulate apically onto older neurons. This neurogenesis depends on Neuregulin 1 type II (NRG1-II)-ErbB signaling. Treatment with an ErbB inhibitor, AG1478 impairs mitoses in the sub-ventricular zone of the optic tectum. Removal of AG1478 resumes sub-ventricular mitoses without precedent mitoses in the apical ventricular zone prior to basal-to-apical accumulation of neurons, suggesting critical roles of ErbB signaling in mitoses for post-mitotic neuron production. Knockdown of NRG1-II impairs both mitoses in the sub-basal/sub-ventricular zone and the ventricular zone. Injection of soluble human NRG1 into the developing brain ameliorates neurogenesis of NRG1-II-knockdown embryos, suggesting a conserved role of NRG1 as a cell-extrinsic signal. From these results, we propose that NRG1-ErbB signaling stimulates cell divisions generating neurons from neural progenitor cells in the developing vertebrate brain.

Immunohistochemical detection of toll-like receptor-2, -4 and -9 in exocrine glands associated with rat alimentary tract.

Localization of Toll-like receptors (TLRs) in the exocrine glands associated with the rat alimentary tract was immunohistochemically studied using anti-TLR antibodies. TLR-2, -4 and -9 were detected in the secretory granules of acinar cells or the luminal substances of the gustatory gland, extraorbital lacrimal gland, Harderian gland, proper gastric gland and pancreas. TLR-2 and -9 were also detected in the mucous acinar cells of the sublingual gland. Positivity for all TLRs was found in the striated borders of columnar epithelial cells and the luminal substances of the intestinal crypts throughout the small intestine, and also in the goblet cells throughout the large intestine. Only TLR-4 was detected in the secretory granules of Paneth cells. A reduction of TLR-4-positive secretory granules and the formation of TLR-4-positive vacuoles were found in the ileal Paneth cells under the hyper-proliferation of indigenous bacteria. In the apical to middle intervillous portions of the ileum, Gram-positive bacterial colonies were significantly more abundant than Gram-negative bacterial colonies, whereas this difference disappeared in the basal intervillous portions. These findings suggest that there are distribution differences in the secretory sources of soluble TLRs that possibly neutralize their luminal ligands, in the rat alimentary tract. Therefore, the bacterial ligand-recognition system composed of the membranous TLRs of villous columnar epithelial cells and soluble TLRs from crypt epithelial cells might contribute to host defense mechanisms for the selective elimination of Gram-positive bacteria rather than Gram-negative bacteria in the rat small intestine.