Requirement for Pdx1 in specification of latent endocrine progenitors in zebrafish

<p>Abstract</p> <p>Background</p> <p>Insulin-producing beta cells emerge during pancreas development in two sequential waves. Recently described later-forming beta cells in zebrafish show high similarity to second wave mammalian beta cells in developmental capacity. Loss-of-function studies in mouse and zebrafish demonstrated that the homeobox transcription factors Pdx1 and Hb9 are both critical for pancreas and beta cell development and discrete stage-specific requirements for these genes have been uncovered. Previously, exocrine and endocrine cell recovery was shown to follow loss of <it>pdx1 </it>in zebrafish, but the progenitor cells and molecular mechanisms responsible have not been clearly defined. In addition, interactions of <it>pdx1 </it>and <it>hb9 </it>in beta cell formation have not been addressed.</p> <p>Results</p> <p>To learn more about endocrine progenitor specification, we examined beta cell formation following morpholino-mediated depletion of <it>pdx1 </it>and <it>hb9</it>. We find that after early beta cell reduction, recovery occurs following loss of either <it>pdx1 </it>or <it>hb9 </it>function. Unexpectedly, simultaneous knockdown of both <it>hb9 </it>and <it>pdx1 </it>leads to virtually complete and persistent beta cell deficiency. We used a <it>NeuroD:EGFP </it>transgenic line to examine endocrine cell behavior <it>in vivo </it>and developed a novel live-imaging technique to document emergence and migration of late-forming endocrine precursors in real time. Our data show that Notch-responsive progenitors for late-arising endocrine cells are predominantly post mitotic and depend on <it>pdx1</it>. By contrast, early-arising endocrine cells are specified and differentiate independent of <it>pdx1</it>.</p> <p>Conclusions</p> <p>The nearly complete beta cell deficiency after combined loss of <it>hb9 </it>and <it>pdx1 </it>suggests functional cooperation, which we clarify as distinct roles in early and late endocrine cell formation. A novel imaging approach permitted visualization of the emergence of late endocrine cells within developing embryos for the first time. We demonstrate a <it>pdx1</it>-dependent progenitor population essential for the formation of duct-associated, second wave endocrine cells. We further reveal an unexpectedly low mitotic activity in these progenitor cells, indicating that they are set aside early in development.</p

Zebrafish hoxd4a Acts Upstream of meis1.1 to Direct Vasculogenesis, Angiogenesis and Hematopoiesis

10.1371/journal.pone.0058857PLoS ONE83

Future directions for therapeutic strategies in post-ischaemic vascularization: a position paper from European Society of Cardiology Working Group on Atherosclerosis and Vascular Biology

Modulation of vessel growth holds great promise for treatment of cardiovascular disease. Strategies to promote vascularization can potentially restore function in ischaemic tissues. On the other hand, plaque neovascularization has been shown to associate with vulnerable plaque phenotypes and adverse events. The current lack of clinical success in regulating vascularization illustrates the complexity of the vascularization process, which involves a delicate balance between pro- and anti-angiogenic regulators and effectors. This is compounded by limitations in the models used to study vascularization that do not reflect the eventual clinical target population. Nevertheless, there is a large body of evidence that validate the importance of angiogenesis as a therapeutic concept. The overall aim of this Position Paper of the ESC Working Group of Atherosclerosis and Vascular biology is to provide guidance for the next steps to be taken from pre-clinical studies on vascularization towards clinical application. To this end, the current state of knowledge in terms of therapeutic strategies for targeting vascularization in post-ischaemic disease is reviewed and discussed. A consensus statement is provided on how to optimize vascularization studies for the identification of suitable targets, the use of animal models of disease, and the analysis of novel delivery methods

Maternal Role Confusion: Relations to Maternal Attachment and Mother-Child Interaction from Infancy to Adolescence

Self-reports of role confusion with the parent in childhood are associated with a variety of adverse outcomes. However, role-confusion has been studied primarily from the point of view of the child. The current study evaluated an instrument for assessing role confusion from maternal interviews rather than from child observations or self-reports in adulthood. Fifty-one mothers participating in a longitudinal study since their own child's infancy were administered the Experiences of Caregiving Interview (C. George & J. Solomon, 1996) when the child was age 20. Interviews were coded using the newly developed Parental Assessment of Role Confusion (PARC; L. Vulliez-Coady & K. Lyons-Ruth, 2009). Maternal PARC scores were related to observational measures of role-confusion in interaction with the child both in infancy and late adolescence. PARC scores also were related to mothers’ hostile-helpless states of mind on the Adult Attachment Interview (C. George, N. Kaplan, & M. Main, 1984, 1985, 1986) and to the extent of Unresolved loss, but not Unresolved Trauma. PARC scores also were related to mothers’ self-reports of helplessness experienced in the parenting role. Discriminant validity of the PARC was demonstrated in that role confusion on the PARC was not related to hostile or disoriented forms of parent–child interaction. Implications for clinical assessment of role confusion are discussed

Endothelial Cell Self-fusion during Vascular Pruning

During embryonic development, vascular networks remodel to meet the increasing demand of growing tissues for oxygen and nutrients. This is achieved by the pruning of redundant blood vessel segments, which then allows more efficient blood flow patterns. Because of the lack of an in vivo system suitable for high-resolution live imaging, the dynamics of the pruning process have not been described in detail. Here, we present the subintestinal vein (SIV) plexus of the zebrafish embryo as a novel model to study pruning at the cellular level. We show that blood vessel regression is a coordinated process of cell rearrangements involving lumen collapse and cell-cell contact resolution. Interestingly, the cellular rearrangements during pruning resemble endothelial cell behavior during vessel fusion in a reversed order. In pruning segments, endothelial cells first migrate toward opposing sides where they join the parental vascular branches, thus remodeling the multicellular segment into a unicellular connection. Often, the lumen is maintained throughout this process, and transient unicellular tubes form through cell self-fusion. In a second step, the unicellular connection is resolved unilaterally, and the pruning cell rejoins the opposing branch. Thus, we show for the first time that various cellular activities are coordinated to achieve blood vessel pruning and define two different morphogenetic pathways, which are selected by the flow environment

Distinct delta and jagged genes control sequential segregation of pancreatic cell types from precursor pools in zebrafish

The different cell types of the vertebrate pancreas arise asynchronously during organogenesis. Beta-cells producing insulin, alpha-cells producing glucagon, and exocrine cells secreting digestive enzymes differentiate sequentially from a common primordium. Notch signaling has been shown to be a major mechanism controlling these cell-fate choices. So far, the pleiotropy of Delta and Jagged/Serrate genes has hindered the evaluation of the roles of specific Notch ligands, as the phenotypes of knock-out mice are lethal before complete pancreas differentiation. Analyses of gene expression and experimental manipulations of zebrafish embryos allowed us to determine individual contributions of Notch ligands to pancreas development. We have found that temporally distinct phases of both endocrine and exocrine cell type specification are controlled by different delta and jagged genes. Specifically, deltaA knock-down embryos lack alpha cells, similarly to mib (Delta ubiquitin ligase) mutants and embryos treated with DAPT, a gamma secretase inhibitor able to block Notch signaling. Conversely, jagged1b morphants develop an excess of alpha-cells. Moreover, the pancreas of jagged2 knock-down embryos has a decreased ratio of exocrine-to-endocrine compartments. Finally, overexpression of Notch1a-intracellular-domain in the whole pancreas primordium or specifically in beta-cells helped us to refine a model of pancreas differentiation in which cells exit the precursor state at defined stages to form the pancreatic cell lineages, and, by a feedback mediated by different Notch ligands, limit the number of other cells that can leave the precursor state

Developmental Role of Zebrafish Protease-Activated Receptor 1 (PAR1) in the Cardio-Vascular System

Thrombin receptor, F2R or PAR1 is a G-protein coupled receptor, located in the membrane of endothelial cells. It has been initially found to transduce signals in hemostasis, but recently also known to act in cancer and in vascular development. Mouse embryos lacking PAR1 function die from hemorrhages with varying frequency at midgestation. We have performed a survey of potential PAR1 homologs in the zebrafish genome and identified a teleost ortholog of mammalian PAR1. Knockdown of par1 function in zebrafish embryos demonstrates a requirement for Par1 in cardio-vascular development. Furthermore, we show that function of Par1 requires the presence of a phylogenetically conserved proteolytic cleavage site and a second intracellular domain. Altogether our results demonstrate a high degree of conservation of PAR1 proteins in the vertebrate lineage in respect to amino acid sequence as well as protein function