Cirone, P., Lin, S., Griesbach, H.L., Yi Zhang, Slusarski D.C. and Crews, C.M. A Role for Planar Cell Polarity Signaling in Angiogenesis. Angiogenesis. 2008; 11(4):347-60.

The planar cell polarity (PCP) pathway is a highly conserved signaling cascade that coordinates both epithelial and axonal morphogenic movements during development. Angiogenesis also involves the growth and migration of polarized cells, although the mechanisms underlying their intercellular communication are poorly understood. Here, using cell culture assays, we demonstrate that inhibition of PCP signaling disrupts endothelial cell growth, polarity, and migration, all of which can be rescued through downstream activation of this pathway by expression of either Daam-1, Diversin or Inversin. Silencing of either Dvl2 or Prickle suppressed endothelial cell proliferation. Moreover, loss of p53 rescues endothelial cell growth arrest but not the migration inhibition caused by PCP disruption. In addition, we show that the zebrafish Wnt5 mutant (pipetail (ppt)), which has impaired PCP signaling, displays vascular developmental defects. These findings reveal a potential role for PCP signaling in the coordinated assembly of endothelial cells into vascular structures and have important implications for vascular remodeling in development and disease.

Ju,R.,Cirone,P.,Lin,S.,Griesbach,H.L.,SlusarskiD.C.andCrews,C.M.Activationofthe Planar Cell Polarity Formin DAAM1 Leads to Inhibition of Endothelial Cell Proliferation, Migration and Angiogenesis. Proceedings of the National Academy of Sciences of the Uni

The Wnt/planar cell polarity (PCP) pathway regulates directed cell movement during development and was recently found to play a critical role in endothelial cell proliferation and angiogenesis [Zhang Y, et al. (2006) Chem Biol 13:1001-1009; Masckauchan TN, et al. (2006) Mol Biol Cell 17:5163-5172]. However, the mechanisms by which PCP signaling components regulate angiogenesis remain unknown. We report that expression of a constitutively active C-terminal domain of Dishevelled-associated activator of morphogenesis 1 (DAAM1) selectively inhibited endothelial cell proliferation. Moreover, this activated construct suppressed endothelial cell migration and the ability to form coordinated networks in vivo and in vitro. Although constitutively active DAAM1 (CDAAM1) induced both actin polymerization and microtubule (MT) stabilization, the stabilization of MTs alone was sufficient to inhibit endothelial cell growth selectively. Inhibition of actin polymerization alone by jasplakinolide treatment failed to reproduce the inhibitory effects of CDAAM1. These results indicate that DAAM1 regulates endothelial cell growth through MT stabilization in a cell type-selective manner and suggest that PCP signaling plays a pivotal role in angiogenesis by regulating MT stabilization.

Schneider, I., Schneider, P.N., Derry, S.W., Lin, S., Barton, L.J., Westfall, T.A., and Slusarski D.C. Zebrafish Nkd1 Promotes Dvl Degradation and is Required for Asymmetric Charon Expression and Left-Right Patterning. Developmental Biology. 2010; 348(1):

The establishment of the left-right (LR) axis in zebrafish embryos relies on signals from the dorsal forerunner cells (DFC) and the Kupffer's vesicle (KV). While the Wnt signaling network influences many aspects of embryonic development, its precise role in LR patterning is still unclear. One branch of the Wnt network leads to stabilization of β-catenin and activation of downstream target genes. Other Wnt ligands appear to act independently of β-catenin to modulate calcium release and influence cell polarity. Central to regulation of β-catenin and coordination of convergent extension (CE) movements is Dishevelled (Dvl). Naked Cuticle (Nkd) binds Dvl and modulates β-catenin-dependent and independent Wnt signaling. Here, we analyze the expression patterns of three zebrafish Nkd homologs and find enriched expression of nkd1 in DFCs and KV. Dvl is degraded upon Nkd1 overexpression in zebrafish. Knockdown of Nkd1 specifically in the DFC results in β-catenin nuclear localization and transcriptional activation as well as alterations to DFC migration, KV formation, ciliogenesis and LR patterning. Furthermore, we identify asymmetric expression of the Nodal antagonist charon around the KV and show that Nkd1 knockdown impacts asymmetric charon expression. Our findings show that Nkd1 acts as a β-catenin antagonist in the DFCs necessary for LR patterning.

Pech, M.F.*, Garbuzov, A.*, Hasegawa, K., Sukhwani, M., Zhang, R.J., Benayoun, B.A., Brockman, S.A., Lin, S., Brunet, A,. Orwig, K.E., and Artandi, S.E. High telomerase is a hallmark of undifferentiated spermatogonia and is required for maintenance of mal

Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter
mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic
population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying
undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated
spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of TertHigh undifferentiated spermatogonia and
TertLow differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular
features of each compartment. Transplantation studies show that germline stem cell activity is confined to the
TertHigh cKit− population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated
spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical
threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells,
thus explaining the broad dependence on telomerase for germline immortality in metazoans.

Chen, L., Roake, C.M., Freund, A., Batista, P.J., Tian, S., Yi, Y.A., Gajera, C.R., Lin, S., Lee, B., Pech, M.F., Venteicher, A.S., Das, R., Chang, H.Y., and Artandi, S.E. An activity switch in human telomerase based on RNA conformation and shaped by TCAB

Ribonucleoprotein enzymes require dynamic conformations of their RNA constituents for regulated catalysis. Human telomerase employs a non-coding RNA (hTR) with a bipartite arrangement of domains-a template-containing core and a distal three-way junction (CR4/5) that stimulates catalysis through unknown means. Here, we show that telomerase activity unexpectedly depends upon the holoenzyme protein TCAB1, which in turn controls conformation of CR4/5. Cells lacking TCAB1 exhibit a marked reduction in telomerase catalysis without affecting enzyme assembly. Instead, TCAB1 inactivation causes unfolding of CR4/5 helices that are required for catalysis and for association with the telomerase reverse-transcriptase (TERT). CR4/5 mutations derived from patients with telomere biology disorders provoke defects in catalysis and TERT binding similar to TCAB1 inactivation. These findings reveal a conformational "activity switch" in human telomerase RNA controlling catalysis and TERT engagement. The identification of two discrete catalytic states for telomerase suggests an intramolecular means for controlling telomerase in cancers and progenitor cells.

Lin, S., Baye, L.M., Westfall, T.A. and Slusarski D.C. Wnt5b-Ryk Pathway Provides Directional Signals to Regulate Gastrulation Movement. Journal of Cell Biology. 2010; 190(2): 263-278.

Noncanonical Wnts are largely believed to act as permissive cues for vertebrate cell movement via Frizzled (Fz). In addition to Fz, Wnt ligands are known to regulate neurite outgrowth through an alternative receptor related to tyrosine kinase (Ryk). However, Wnt–Ryk signaling during embryogenesis is less well characterized. In this study, we report a role for Wnt5b as an instructive cue to regulate gastrulation movements through Ryk. In zebrafish, Ryk deficiency impairs Wnt5b-induced Ca2+ activity and directional cell movement. Wnt5b–Ryk signaling promotes polarized cell protrusions. Upon Wnt5b stimulation, Fz2 but not Ryk recruits Dishevelled to the cell membrane, suggesting that Fz2 and Ryk mediate separate pathways. Using co-culture assays to generate directional Wnt5b cues, we demonstrate that Ryk-expressing cells migrate away from the Wnt5b source. We conclude that full-length Ryk conveys Wnt5b signals in a directional manner during gastrulation.