Prohormone convertase 1/3 deficiency causes obesity due to impaired proinsulin processing

Defective insulin processing is associated with obesity and diabetes. Prohormone convertase 1/3 (PC1/3) is an endopeptidase required for the processing of neurotransmitters and hormones. PC1/3 deficiency and genome-wide association studies relate PC1/3 with early onset obesity. Here, we find that deletion of PC1/3 in obesity-related neuronal cells expressing proopiomelanocortin mildly and transiently change body weight and fail to produce a phenotype when targeted to Agouti-related peptide- or nestin-expressing tissues. In contrast, pancreatic β cell-specific PC1/3 ablation induces hyperphagia with consecutive obesity despite uncontrolled diabetes with glucosuria. Obesity develops not due to impaired pro-islet amyloid polypeptide processing but due to impaired insulin maturation. Proinsulin crosses the blood-brain-barrier but does not induce central satiety. Accordingly, insulin therapy prevents hyperphagia. Further, islet PC1/3 expression levels negatively correlate with body mass index in humans. In this work, we show that impaired PC1/3-mediated proinsulin processing, as observed in human prediabetes, promotes hyperphagic obesity

Junction-based lamellipodia drive endothelial cell rearrangements in vivo via a VE-cadherin-F-actin based oscillatory cell-cell interaction

Angiogenesis and vascular remodeling are driven by extensive endothelial cell movements. Here, we present in vivo evidence that endothelial cell movements are associated with oscillating lamellipodia-like structures, which emerge from cell junctions in the direction of cell movements. High-resolution time-lapse imaging of these junction-based lamellipodia (JBL) shows dynamic and distinct deployment of junctional proteins, such as F-actin, VE-cadherin and ZO1, during JBL oscillations. Upon initiation, F-actin and VE-cadherin are broadly distributed within JBL, whereas ZO1 remains at cell junctions. Subsequently, a new junction is formed at the front of the JBL, which then merges with the proximal junction. Rac1 inhibition interferes with JBL oscillations and disrupts cell elongation-similar to a truncation in ve-cadherin preventing VE-cad/F-actin interaction. Taken together, our observations suggest an oscillating ratchet-like mechanism, which is used by endothelial cells to move over each other and thus provides the physical means for cell rearrangements

"In vivo" analysis of junctional dynamics underlying angiogenic cell behaviors

The blood circulatory system delivers nutrients and oxygen to tissues and organs. Moreover, the circulatory system retains high plasticity throughout an organism’s life, which is important for physiological processes, such as wound healing, but also during pathological processes, such as tumor growth. Blood vessels can sprout from existing ones, a process called angiogenesis, to create a more branched network that reaches avascular and the most distal parts of an animal body. The morphogenetic processes that underlie angiogenesis can be studied at cellular resolution in the zebrafish embryo. Moreover, the fast development of the embryo allows to follow these processes in real-time. In the trunk, where metameric vessels sprout from the dorsal aorta, cells migrate collectively in a hierarchy defined by a leading endothelial cell at the tip of the sprout and several following cells, called stalk cells. Once the tip cells reach the level of the dorsal neural tube, they extend laterally and initiate the anastomosis process with tip cells from the neighboring segments. Eventually, cells rearrange and form a patent lumen, which allows blood circulation. Angiogenic sprout outgrowth and anastomosis involve complex cell behaviors and cell-cell interactions, which need to be precisely orchestrated. The molecular mechanisms underlying these cellular activities are not known. However, proteins that mediate endothelial specific cell adhesion are good candidates to promote concerted cell behaviors. The goal of my thesis was (i) to characterize the cell shape changes that occur during angiogenic sprouting, (ii) to analyze the function of VE-cadherin in this process and (iii) to analyze the role of VE-cadherin and Esama, as candidate proteins, in the initiation of endothelial cell-cell interaction at the onset of vascular anastomosis. I found that two major cell behaviors contribute to angiogenic sprouting. While cell migration is predominantly used by the tip cell, elongation of the stalk is mainly achieved by extensive stalk cell elongation, rather than by pulling forces exerted by the tip cell. VE-cadherin, which is the major component of adherens junction of endothelial cells, is required for concerted angiogenic cell junctional elongation. The absence of VE-cadherin in stalk cells leads to a disorganized cortical F-actin network, which reflects the elongation defects. Furthermore, the loss of VE-cadherin function can be phenocopied by inhibiting actin polymerization. Anastomosis is initiated by filopodial contacts between endothelial cells. The formation of these contacts is thought to be mediated by endothelial specific adhesion molecules, which provide adhesion as well as cell type specificity. We have previously shown that VE-cadherin plays an important role in anastomotic contact formation. However, our observation that tip cells can still generate contacts in the absence of VE-cadherin, prompted us to investigate the role of a second endothelial-specific adhesion molecule, Esama, during anastomosis. In my thesis I generated a targeted mutation in the zebrafish esama gene using TALEN technology and started to analyze the loss of function. Embryos mutant for esama are viable and do not show major vascular defects, except for small, transient gaps in junctional rings. However, zebrafish embryos lacking both, VE-cadherin and Esama, show frequent detachments of stalk from tip cells, ineffective cell-type specific recognition and strongly protrusive cell morphologies. All together, the phenotypes of the ve-cadherin mutants are aggravated by the simultaneous absence of Esama. Moreover, the double mutant shows junctional discontinuities, seen as big gaps within the junctional rings between stalk cells. Our results support a model for angiogenic sprout elongation by cell shape changes orchestrated by VE-cadherin. VE-cadherin connects the actin cytoskeletons of neighboring stalk cells and drives the cell elongation by localized actin polymerization at the edges of the elongating junctions. Esama and VE-cadherin have partly overlapping functions during angiogenic sprouting and anastomosis. Both proteins are required for endothelial contact formation during anastomosis, but also for the maintenance of structural integrity during angiogenic sprouting

Endothelial cell division in angiogenic sprouts of differing cellular architecture

The vasculature of the zebrafish trunk is composed of tubes with different cellular architectures. Unicellular tubes form their lumen through membrane invagination and transcellular cell hollowing, whereas multicellular vessels become lumenized through a chord hollowing process. Endothelial cell proliferation is essential for the subsequent growth and maturation of the blood vessels. However, how cell division, lumen formation and cell rearrangement are coordinated during angiogenic sprouting has so far not been investigated at detailed cellular level. Reasoning that different tubular architectures may impose discrete mechanistic constraints on endothelial cell division, we analyzed and compared the sequential steps of cell division, namely mitotic rounding, cytokinesis, actin re-distribution and adherence junction formation, in different blood vessels. In particular, we characterized the interplay between cell rearrangement, mitosis and lumen dynamics within unicellular and multicellular tubes. The lumen of unicellular tubes becomes constricted and is ultimately displaced from the plane of cell division, where a de novo junction forms through the recruitment of junctional proteins at the site of abscission. By contrast, the new junctions separating the daughter cells within multicellular tubes form through the alteration of pre-existing junctions, and the lumen is retained throughout mitosis. We also describe variations in the progression of cytokinesis: while membrane furrowing between daughter cells is symmetric in unicellular tubes, we found that it is asymmetric in those multicellular tubes that contained a taut intercellular junction close to the plane of division. Our findings illustrate that during the course of normal development, the cell division machinery can accommodate multiple tube architectures, thereby avoiding disruptions to the vascular network

Distinct and redundant functions of Esam and VE-cadherin during vascular morphogenesis

The cardiovascular system forms during early embryogenesis and adapts to embryonic growth by sprouting angiogenesis and vascular remodeling. These processes require fine-tuning of cell-cell adhesion to maintain and reestablish endothelial contacts, while allowing cell motility. We have compared the contribution of two endothelial cell specific adhesion proteins - VE-cadherin (VE-cad/Cdh5) and Esama (Endothelial cell-selective adhesion molecule a) - during angiogenic sprouting and blood vessel fusion (anastomosis) in the zebrafish embryo by genetic analyses. Different combinations of mutant alleles can be placed into a phenotypic series with increasing defects in filopodial contact formation. Contact formation in esama mutants appear wild-type like, while esama(-/-); ve-cad(+/-)and ve-cad single mutants exhibit intermediate phenotypes. The lack of both proteins interrupts filopodial interaction completely. Furthermore, double mutants do not form a stable endothelial monolayer, display intrajunctional gaps, dislocalization of Zo-1 and defects in apical-basal polarization. In summary, VE-cadherin and Esama have distinct and redundant functions during blood vessel morphogenesis and both adhesion proteins are central to endothelial cell recognition during anastomosis

Modeling hematopoietic disorders in zebrafish

Zebrafish offer a powerful vertebrate model for studies of development and disease. The major advantages of this model include the possibilities of conducting reverse and forward genetic screens and of observing cellular processes by; in vivo; imaging of single cells. Moreover, pathways regulating blood development are highly conserved between zebrafish and mammals, and several discoveries made in fish were later translated to murine and human models. This review and accompanying poster provide an overview of zebrafish hematopoiesis and discuss the existing zebrafish models of blood disorders, such as myeloid and lymphoid malignancies, bone marrow failure syndromes and immunodeficiencies, with a focus on how these models were generated and how they can be applied for translational research

Cdh5/VE-cadherin Promotes Endothelial Cell Interface Elongation via Cortical Actin Polymerization during Angiogenic Sprouting

SummaryOrgan morphogenesis requires the coordination of cell behaviors. Here, we have analyzed dynamic endothelial cell behaviors underlying sprouting angiogenesis in vivo. Two different mechanisms contribute to sprout outgrowth: tip cells show strong migratory behavior, whereas extension of the stalk is dependent upon cell elongation. To investigate the function of Cdh5 in sprout outgrowth, we generated null mutations in the zebrafish cdh5 gene, and we found that junctional remodeling and cell elongation are impaired in mutant embryos. The defects are associated with a disorganization of the actin cytoskeleton and cannot be rescued by expression of a truncated version of Cdh5. Finally, the defects in junctional remodeling can be phenocopied by pharmacological inhibition of actin polymerization, but not by inhibiting actin-myosin contractility. Taken together, our results support a model in which Cdh5 organizes junctional and cortical actin cytoskeletons, as well as provides structural support for polymerizing F-actin cables during endothelial cell elongation

Junction-based lamellipodia drive endothelial cell arrangements in vivo via a VE-cadherin/F-actin based oscillatory ratchet mechanism

Angiogenesis and vascular remodeling are driven by a wide range of endothelial cell behaviors, such as cell divisions, cell movements, cell shape and polarity changes. To decipher the cellular and molecular mechanism of cell movements, we have analyzed the dynamics of different junctional components during blood vessel anastomosis in vivo. We show that endothelial cell movements are associated with oscillating lamellipodia-like structures, which are orientated in the direction of these movements. These structures emerge from endothelial cell junctions and we thus call them junction-based lamellipodia (JBL). High-resolution time-lapse imaging shows that JBL are formed by F-actin based protrusions at the front end of moving cells. These protrusions also contain diffusely distributed VE-cadherin, whereas the junctional protein ZO-1 (Zona occludens 1) remains at the junction. Subsequently, a new junction is formed at the front of the JBL and the proximal junction is pulled towards the newly established distal junction. JBL function is highly dependent on F-actin dynamics. Inhibition of F-actin polymerization prevents JBL formation, whereas Rac-1 inhibition interferes with JBL oscillations. Both interventions disrupt endothelial junction formation and cell elongation. To examine the role of VE-cadherin (encoded by cdh5 gene) in this process, we generated a targeted mutation in VE-cadherin gene (cdh5ubs25), which prevents VE-cad/F-actin interaction. Although homozygous ve-cadherin mutants form JBL, these JBL are less dynamic and do not promote endothelial cell elongation. Taken together, our observations suggest a novel oscillating ratchet-like mechanism, which is used by endothelial cells to move along or over each other and thus provides the physical means for cell rearrangements

The evolution of cichlid fish egg-spots is linked with a cis-regulatory change

The origin of novel phenotypic characters is a key component in organismal diversification; yet, the mechanisms underlying the emergence of such evolutionary novelties are largely unknown. Here we examine the origin of egg-spots, an evolutionary innovation of the most species-rich group of cichlids, the haplochromines, where these conspicuous male fin colour markings are involved in mating. Applying a combination of RNAseq, comparative genomics and functional experiments, we identify two novel pigmentation genes, fhl2a and fhl2b, and show that especially the more rapidly evolving b-paralog is associated with egg-spot formation. We further find that egg-spot bearing haplochromines, but not other cichlids, feature a transposable element in the cis-regulatory region of fhl2b. Using transgenic zebrafish, we finally demonstrate that this region shows specific enhancer activities in iridophores, a type of pigment cells found in egg-spots, suggesting that a cis-regulatory change is causally linked to the gain of expression in egg-spot bearing haplochromines