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• W2088903662 abstract "Studies have attributed several functions to the Eaf family, including tumor suppression and eye development. Given the potential association between cancer and development, we set forth to explore Eaf1 and Eaf2/U19 activity in vertebrate embryogenesis, using zebrafish. In situ hybridization revealed similar eaf1 and eaf2/u19 expression patterns. Morpholino-mediated knockdown of either eaf1 or eaf2/u19 expression produced similar morphological changes that could be reversed by ectopic expression of target or reciprocal-target mRNA. However, combination of Eaf1 and Eaf2/U19 (Eafs)-morpholinos increased the severity of defects, suggesting that Eaf1 and Eaf2/U19 only share some functional redundancy. The Eafs knockdown phenotype resembled that of embryos with defects in convergence and extension movements. Indeed, knockdown caused expression pattern changes for convergence and extension movement markers, whereas cell tracing experiments using kaeda mRNA showed a correlation between Eafs knockdown and cell migration defects. Cardiac and pancreatic differentiation markers revealed that Eafs knockdown also disrupted midline convergence of heart and pancreatic organ precursors. Noncanonical Wnt signaling plays a key role in both convergence and extension movements and midline convergence of organ precursors. We found that Eaf1 and Eaf2/U19 maintained expression levels of wnt11 and wnt5. Moreover, wnt11 or wnt5 mRNA partially rescued the convergence and extension movement defects occurring in eafs morphants. Wnt11 and Wnt5 converge on rhoA, so not surprisingly, rhoA mRNA more effectively rescued defects than either wnt11 or wnt5 mRNA alone. However, the ectopic expression of wnt11 and wnt5 did not affect eaf1 and eaf2/u19 expression. These data indicate that eaf1 and eaf2/u19 act upstream of noncanonical Wnt signaling to mediate convergence and extension movements. Studies have attributed several functions to the Eaf family, including tumor suppression and eye development. Given the potential association between cancer and development, we set forth to explore Eaf1 and Eaf2/U19 activity in vertebrate embryogenesis, using zebrafish. In situ hybridization revealed similar eaf1 and eaf2/u19 expression patterns. Morpholino-mediated knockdown of either eaf1 or eaf2/u19 expression produced similar morphological changes that could be reversed by ectopic expression of target or reciprocal-target mRNA. However, combination of Eaf1 and Eaf2/U19 (Eafs)-morpholinos increased the severity of defects, suggesting that Eaf1 and Eaf2/U19 only share some functional redundancy. The Eafs knockdown phenotype resembled that of embryos with defects in convergence and extension movements. Indeed, knockdown caused expression pattern changes for convergence and extension movement markers, whereas cell tracing experiments using kaeda mRNA showed a correlation between Eafs knockdown and cell migration defects. Cardiac and pancreatic differentiation markers revealed that Eafs knockdown also disrupted midline convergence of heart and pancreatic organ precursors. Noncanonical Wnt signaling plays a key role in both convergence and extension movements and midline convergence of organ precursors. We found that Eaf1 and Eaf2/U19 maintained expression levels of wnt11 and wnt5. Moreover, wnt11 or wnt5 mRNA partially rescued the convergence and extension movement defects occurring in eafs morphants. Wnt11 and Wnt5 converge on rhoA, so not surprisingly, rhoA mRNA more effectively rescued defects than either wnt11 or wnt5 mRNA alone. However, the ectopic expression of wnt11 and wnt5 did not affect eaf1 and eaf2/u19 expression. These data indicate that eaf1 and eaf2/u19 act upstream of noncanonical Wnt signaling to mediate convergence and extension movements. EAF1 (ELL-associated factor 1) was first discovered through its ability to associate with the protein ELL (eleven-nineteen lysine-rich leukemia), a fusion partner of MLL in the t(11;19)(q23;p13.1) chromosomal translocation associated with acute myeloid leukemia (1Thirman M.J. Levitan D.A. Kobayashi H. Simon M.C. Rowley J.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12110-12114Crossref PubMed Scopus (202) Google Scholar, 2Simone F. Polak P.E. Kaberlein J.J. Luo R.T. Levitan D.A. Thirman M.J. Blood. 2001; 98: 201-209Crossref PubMed Scopus (61) Google Scholar). Subsequent studies found a second binding partner for ELL, EAF2, which was independently identified as an androgen up-regulated gene in the rat prostate and named human up-regulated 19 (U19) (3Wang Z. Tufts R. Haleem R. Cai X. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12999-13004Crossref PubMed Scopus (93) Google Scholar, 4Simone F. Luo R.T. Polak P.E. Kaberlein J.J. Thirman M.J. Blood. 2003; 101: 2355-2362Crossref PubMed Scopus (62) Google Scholar). ELL binds to RNA polymerase II and acts as a transcriptional elongation factor whose targeted deletion leads to embryonic lethality in mice (5Shilatifard A. Lane W.S. Jackson K.W. Conaway R.C. Conaway J.W. Science. 1996; 271: 1873-1876Crossref PubMed Scopus (279) Google Scholar, 6Mitani K. Yamagata T. Iida C. Oda H. Maki K. Ichikawa M. Asai T. Honda H. Kurokawa M. Hirai H. Biochem. Biophys. Res. Commun. 2000; 279: 563-567Crossref PubMed Scopus (22) Google Scholar). Both EAF1 and EAF2, which share significant sequence homology, stimulate ELL elongation activity (7Kong S.E. Banks C.A. Shilatifard A. Conaway J.W. Conaway R.C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10094-10098Crossref PubMed Scopus (69) Google Scholar). Studies by Luo et al. (8Luo R.T. Lavau C. Du C. Simone F. Polak P.E. Kawamata S. Thirman M.J. Mol. Cell. Biol. 2001; 21: 5678-5687Crossref PubMed Scopus (70) Google Scholar) argued that EAF proteins are important in MLL-ELL leukemogenesis, whereas our previous studies showed that EAF2/U19 inhibits xenograft prostate tumor growth and undergoes down-regulation in prostate cancer cell lines (9Xiao W. Zhang Q. Jiang F. Pins M. Kozlowski J.M. Wang Z. Cancer Res. 2003; 63: 4698-4704PubMed Google Scholar). These findings link the EAF2/U19 gene with two major human cancers: prostate cancer and acute myeloid leukemia. To investigate the function EAF2/U19 in vivo, we constructed a murine knock-out model. The EAF2/U19 knock-out mice develop B-cell lymphoma, lung adenocarcinoma, hepatocellular carcinoma, and prostate intraepithelial neoplasia with high frequency (10Xiao W. Zhang Q. Habermacher G. Yang X. Zhang A.Y. Cai X. Hahn J. Liu J. Pins M. Doglio L. Dhir R. Gingrich J. Wang Z. Oncogene. 2008; 27: 1536-1544Crossref PubMed Scopus (52) Google Scholar). In addition, we demonstrated that EAF2/U19 could bind to and stabilize the classic tumor suppressor-pVHL (11Xiao W. Ai J. Habermacher G. Volpert O. Yang X. Zhang A.Y. Hahn J. Cai X. Wang Z. Cancer Res. 2009; 69: 2599-2606Crossref PubMed Scopus (26) Google Scholar). These findings further support EAF2/U19 as a potential tumor suppressor; however, the molecular mechanisms underlying this activity remain poorly defined.Evidence suggests that oncogenes and tumor suppressors may play a role in normal vertebrate embryogenesis. Thus, the evaluation of how these proteins function during embryogenesis would not only lead to a better understanding of development but may also shed light on how these proteins contribute to tumor initiation and progression (12Lee H. Kimelman D. Dev. Cell. 2002; 2: 607-616Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 13Langheinrich U. Hennen E. Stott G. Vacun G. Curr. Biol. 2002; 12: 2023-2028Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Several studies have shown that eaf2/u19 appears to be important during embryogenesis, particularly in eye development (14Li M. Wu X. Zhuang F. Jiang S. Jiang M. Liu Y.H. Dev. Dyn. 2003; 228: 273-280Crossref PubMed Scopus (19) Google Scholar, 15Maurus D. Héligon C. Bürger-Schwärzler A. Brändli A.W. Kühl M. EMBO J. 2005; 24: 1181-1191Crossref PubMed Scopus (86) Google Scholar), but its function in vertebrate embryogenesis remains unclear.In vertebrates, the body plan is established during gastrulation by a set of morphogenetic processes, including epiboly, internalization, and convergence and extension movements (16Myers D.C. Sepich D.S. Solnica-Krezel L. Trends Genet. 2002; 18: 447-455Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Gastrulation starts with the epiboly of the enveloping layer and deep cells and the internalization of prospective mesendodermal cells, followed by convergence and extension movements. convergence and extension movements narrow embryonic tissues mediolaterally and lengthen them anteroposteriorly (17Keller R. Shih J. Domingo C. Dev. Suppl. 1992; : 81-91PubMed Google Scholar). By convergence movements, the precursor cells of many organs migrate toward midline from bilateral tissues and assemble to form the primitive organs (18Stainier D.Y. Nat. Rev. 2001; 2: 39-48Crossref Scopus (321) Google Scholar, 19Ober E.A. Olofsson B. Mäkinen T. Jin S.W. Shoji W. Koh G.Y. Alitalo K. Stainier D.Y. EMBO Rep. 2004; 5: 78-84Crossref PubMed Scopus (86) Google Scholar). During the convergence and extension movements, multiple signaling cascades control coordinated cell behaviors. silberblick/wnt11 (slb) and pipetail/wnt5 (ppt) zebrafish mutants show abnormal convergence and extension movements and display a short tail and body axis (20Heisenberg C.P. Tada M. Rauch G.J. Saúde L. Concha M.L. Geisler R. Stemple D.L. Smith J.C. Wilson S.W. Nature. 2000; 405: 76-81Crossref PubMed Scopus (826) Google Scholar, 21Kilian B. Mansukoski H. Barbosa F.C. Ulrich F. Tada M. Heisenberg C.P. Mech. Dev. 2003; 120: 467-476Crossref PubMed Scopus (258) Google Scholar). Thus, the noncanonical Wnt cascade has been proposed to be essential for convergence and extension movements. In addition, rhoA has been reported to act downstream of wnt5 and wnt11 to regulate convergence and extension movements (22Zhu S. Liu L. Korzh V. Gong Z. Low B.C. Cell. Signal. 2006; 18: 359-372Crossref PubMed Scopus (92) Google Scholar).We employed the zebrafish embryo as a model system to investigate the role of eaf1 and eaf2/u19 in vertebrate development. Based on initial findings, we focused our examination on the regulation of convergence and extension movements and midline convergence of the heart and pancreas primordia by Eaf1 and Eaf2/U19 during zebrafish embryogenesis. We also examined whether eaf1 and eaf2/u19 regulate convergence and extension movements by noncanonical Wnt signaling.DISCUSSIONPrevious studies have established that EAF2/U19 contributes to tumor suppression (9Xiao W. Zhang Q. Jiang F. Pins M. Kozlowski J.M. Wang Z. Cancer Res. 2003; 63: 4698-4704PubMed Google Scholar, 10Xiao W. Zhang Q. Habermacher G. Yang X. Zhang A.Y. Cai X. Hahn J. Liu J. Pins M. Doglio L. Dhir R. Gingrich J. Wang Z. Oncogene. 2008; 27: 1536-1544Crossref PubMed Scopus (52) Google Scholar). Other studies suggest that EAF1 and EAF2/U19, like other tumor suppressors, may play a role in vertebrate development (14Li M. Wu X. Zhuang F. Jiang S. Jiang M. Liu Y.H. Dev. Dyn. 2003; 228: 273-280Crossref PubMed Scopus (19) Google Scholar, 15Maurus D. Héligon C. Bürger-Schwärzler A. Brändli A.W. Kühl M. EMBO J. 2005; 24: 1181-1191Crossref PubMed Scopus (86) Google Scholar). However, their exact function in embryogenesis remains unclear. To better understand the cellular function of EAF1 and EAF2/U19 and subsequently how these proteins suppress tumors will require a more extensive investigation of their roles in embryogenesis. Here, we are the first to present data showing that eaf1 and eaf2/u19 mediate effective convergence and extension movements through the maintenance of wnt11 and wnt5 expression during zebrafish gastrulation.Eaf family members demonstrated a high degree of conservation across species and with each other, so not surprisingly, the expression of eaf1 and eaf2/u19 observed in the zebrafish mirrored the pattern seen in the mouse (14Li M. Wu X. Zhuang F. Jiang S. Jiang M. Liu Y.H. Dev. Dyn. 2003; 228: 273-280Crossref PubMed Scopus (19) Google Scholar). However, intercrosses of EAF2/U19 heterozygous knock-out mice yielded Eaf2/U19-null offspring at Mendelian ratios at birth, suggesting that early mouse development did not require EAF2/U19 (10Xiao W. Zhang Q. Habermacher G. Yang X. Zhang A.Y. Cai X. Hahn J. Liu J. Pins M. Doglio L. Dhir R. Gingrich J. Wang Z. Oncogene. 2008; 27: 1536-1544Crossref PubMed Scopus (52) Google Scholar). Eaf2/U19 knockdown in zebrafish embryos alone caused defects in convergence and extension movements in our study. This phenotypic difference between mouse and zebrafish might result from either the redundant function of EAF1 and EAF2/U19 in mouse embryogenesis or from slightly different functions of EAF2/U19 in mammalians and lower vertebrate fish. Indeed, functional differences between mammalian and fish genes are not uncommon (36Correa R.G. Matsui T. Tergaonkar V. Rodriguez-Esteban C. Izpisua-Belmonte J.C. Verma I.M. Curr. Biol. 2005; 15: 1291-1295Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In addition, Eaf2/U19 knockdown in Xenopus laevis caused defects in eye development, which was a pure eye phenotype unrelated to the defect of convergence and extension movement (15Maurus D. Héligon C. Bürger-Schwärzler A. Brändli A.W. Kühl M. EMBO J. 2005; 24: 1181-1191Crossref PubMed Scopus (86) Google Scholar). However, eaf2/u19 knockdown in zebrafish caused shorter eye distance or eye fusion (partial cyclopia) resulting from the defect in convergence and extension movements (Fig. 8C). Together, eaf1 and eaf2/u19 might play different roles in eye development during embryogenesis. Further demonstrating the different mechanism of eaf1 and eaf2/u19 playing their roles in eye formation among species probably will give us a more complete picture about the function of eaf1 and eaf2/u19 in embryogenesis.eaf1 and eaf2/u19 appeared to play partially redundant roles in the regulation of convergence and extension movements. Knockdown of both eaf1 and eaf2/u19 resulted in abnormal cell behavior in both the head and trunk mesendoderm (Fig. 2). The phenotypes observed in eafs morphants included disorganized head structure, reduced body axis, and shorter tail and were similar to those seen in embryos with a disruption of convergence and extension movements (20Heisenberg C.P. Tada M. Rauch G.J. Saúde L. Concha M.L. Geisler R. Stemple D.L. Smith J.C. Wilson S.W. Nature. 2000; 405: 76-81Crossref PubMed Scopus (826) Google Scholar, 21Kilian B. Mansukoski H. Barbosa F.C. Ulrich F. Tada M. Heisenberg C.P. Mech. Dev. 2003; 120: 467-476Crossref PubMed Scopus (258) Google Scholar, 33Wallingford J.B. Fraser S.E. Harland R.M. Dev. Cell. 2002; 2: 695-706Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 37Yamashita S. Miyagi C. Carmany-Rampey A. Shimizu T. Fujii R. Schier A.F. Hirano T. Dev. Cell. 2002; 2: 363-375Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 38Topczewski J. Sepich D.S. Myers D.C. Walker C. Amores A. Lele Z. Hammerschmidt M. Postlethwait J. Solnica-Krezel L. Dev. Cell. 2001; 1: 251-264Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 39Jessen J.R. Topczewski J. Bingham S. Sepich D.S. Marlow F. Chandrasekhar A. Solnica-Krezel L. Nat. Cell Biol. 2002; 4: 610-615Crossref PubMed Scopus (396) Google Scholar). On the basis of phenotype, the marker gene expression patterns, and cell tracing experiments, we concluded that eaf1 and eaf2/u19 served as novel regulators of convergence and extension movements (FIGURE 2, FIGURE 3, FIGURE 4). In reciprocal rescue experiments, eaf1 mRNA could rescue Eaf2-MO1 knockdowns, and eaf2/u19 mRNA could rescue Eaf1-MO knockdowns, suggesting redundant roles for Eaf1 and Eaf2/U19 in the regulation of convergence and extension movements. However, knockdown of either eaf1 or eaf2/u19 produced similar defects that increased in severity with the combined knockdown of both Eaf1 and Eaf2/U19 (FIGURE 2, FIGURE 3, FIGURE 4), indicating that these proteins do not have completely redundant functions.In this study, we found that eaf1 and eaf2/u19 might contribute to the regulation of convergence and extension movements through noncanonical Wnt signaling. Eaf1 and Eaf2/U19 knockdown dramatically down-regulated wnt11 expression, whereas introduction of ectopic eaf1 and eaf2/u19 mRNA increased wnt11 expression (Fig. 7). Moreover, wnt11 mRNA rescued convergence and extension movement defects caused by Eaf1 and Eaf2/U19 knockdown with high frequency (Fig. 9A). However, overexpression of wnt11 by mRNA injection did not affect the expression of eaf1 and eaf2/u19 (Fig. 8). These findings imply that eaf1 and eaf2/u19 act upstream of wnt11 to control its expression and to govern convergence and extension movements. Furthermore, Eaf1 and Eaf2/U19 knockdown down-regulated wnt5 expression (Fig. 7, A and C), and wnt5 mRNA, in turn, also rescued defects in convergence and extension movements caused by the loss of eaf1 and eaf2/u19 (Fig. 9A (A4)). However, overexpression of eaf1 and eaf2/u19 also down-regulated wnt5 expression, with expression levels comparable with those seen in the eafs morphants (Fig. 7, E and G). This suggests that the maintenance of wnt5 might require a specific level or ratio of eaf1 and eaf2/u19. Similar to that of wnt11, wnt5 overexpression did not affect the expression of eaf1 and eaf2/u19, suggesting that eaf1 and eaf2/u19 also act upstream of wnt5. As a downstream activator of noncanonical Wnt ligands, RhoA rescued convergence and extension movement defect more effectively than Wnt5 and Wnt11 (Fig. 8), indicating a convergence of wnt5 and wnt11 on rhoA during the regulation of convergence and extension movements. Of note, the expression patterns of eaf1 and eaf2/u19 were similar to that of rhoA during zebrafish and Xenopus embryogenesis (22Zhu S. Liu L. Korzh V. Gong Z. Low B.C. Cell. Signal. 2006; 18: 359-372Crossref PubMed Scopus (92) Google Scholar, 40Wünnenberg-Stapleton K. Blitz I.L. Hashimoto C. Cho K.W. Development. 1999; 126: 5339-5351Crossref PubMed Google Scholar).Notably, eaf1 and eaf2/u19 have activity in stimulating ELL transcriptional elongation (7Kong S.E. Banks C.A. Shilatifard A. Conaway J.W. Conaway R.C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10094-10098Crossref PubMed Scopus (69) Google Scholar). Thus, if knockdown of eaf1 and eaf2/u19 only caused inhibition of gene expression, eafs regulating the expression of Wnts (wnt11, wnt5, and wnt11r) presented in this study might be nonspecific as a result of disrupting the general function of eafs as regulators of transcriptional elongation. However, in addition to wnt4a,4 foxo5 (Fig. 9A) as well as other genes5 were up-regulated by eafs knockdown, unrelated to their function as regulators of transcriptional elongation. Therefore, eafs knockdown inhibiting the expression of Wnts appears to be specific. Taking into consideration that the knockdown of eafs did not affect cell fate specification at the beginning of gastrulation, it suggests that eafs might directly regulate noncanonical Wnt ligands (especially for wnt11). However, this conclusion needs to be further verified by more direct ways, such as promoter chromatin immunoprecipitation assays, etc.Knockdown of Eaf1 and Eaf2/U19 protein levels also resulted in the failure of fusion of the heart and pancreas primordia (Fig. 6), but neither endoderm fate determination genes nor myocardial precursor marker genes displayed significant changes in their expression levels (Fig. 6, F–H). These observations imply that eaf1 and eaf2/u19 act as novel regulators of midline convergence of both endoderm- and mesoderm-derived organ primordia without affecting the specification of progenitors.Although a primary role of non-canonical Wnt signaling was to govern convergence and extension movements, studies had also shown that the three Wnt noncanonical ligands, wnt4a, wnt11, and wnt11r, redundantly regulated midline convergence of organ primordia, in zebrafish embryos (31Matsui T. Raya A. Kawakami Y. Callol-Massot C. Capdevila J. Rodríguez-Esteban C. Izpisúa Belmonte J.C. Genes Dev. 2005; 19: 164-175Crossref PubMed Scopus (133) Google Scholar, 41Miyasaka K.Y. Kida Y.S. Sato T. Minami M. Ogura T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11274-11279Crossref PubMed Scopus (33) Google Scholar). In this study, we showed that the midline migration of heart precursors and pancreas precursors required expression of eaf1 and eaf2/u19 (Fig. 6). We also showed that eaf1 and eaf2/u19 mediated midline convergence through noncanonical Wnt signaling, specifically by regulating expression of wnt11 and wnt11r (). Unexpectedly, however, knockdown of eaf1 and eaf2/u19 up-regulated wnt4a expression.4 This observation suggested that, unlike wnt11 and wntllr, wnt4a was not a downstream factor of eaf1 and eaf2/u19 in the signaling pathway that regulated midline convergence of organ precursors.In conclusion, our data are the first to demonstrate that eaf1 and eaf2/u19 have essential roles in regulating embryonic cell behavior and migration. Although the complete pathway has yet to be defined, our study shows that eaf1 and eaf2/u19 function in this pathway by specifically modulating expression of wnt11 and wnt5, which in turn converge on rhoA for the positive regulation of convergence and extension movements. EAF1 (ELL-associated factor 1) was first discovered through its ability to associate with the protein ELL (eleven-nineteen lysine-rich leukemia), a fusion partner of MLL in the t(11;19)(q23;p13.1) chromosomal translocation associated with acute myeloid leukemia (1Thirman M.J. Levitan D.A. Kobayashi H. Simon M.C. Rowley J.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12110-12114Crossref PubMed Scopus (202) Google Scholar, 2Simone F. Polak P.E. Kaberlein J.J. Luo R.T. Levitan D.A. Thirman M.J. Blood. 2001; 98: 201-209Crossref PubMed Scopus (61) Google Scholar). Subsequent studies found a second binding partner for ELL, EAF2, which was independently identified as an androgen up-regulated gene in the rat prostate and named human up-regulated 19 (U19) (3Wang Z. Tufts R. Haleem R. Cai X. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12999-13004Crossref PubMed Scopus (93) Google Scholar, 4Simone F. Luo R.T. Polak P.E. Kaberlein J.J. Thirman M.J. Blood. 2003; 101: 2355-2362Crossref PubMed Scopus (62) Google Scholar). ELL binds to RNA polymerase II and acts as a transcriptional elongation factor whose targeted deletion leads to embryonic lethality in mice (5Shilatifard A. Lane W.S. Jackson K.W. Conaway R.C. Conaway J.W. Science. 1996; 271: 1873-1876Crossref PubMed Scopus (279) Google Scholar, 6Mitani K. Yamagata T. Iida C. Oda H. Maki K. Ichikawa M. Asai T. Honda H. Kurokawa M. Hirai H. Biochem. Biophys. Res. Commun. 2000; 279: 563-567Crossref PubMed Scopus (22) Google Scholar). Both EAF1 and EAF2, which share significant sequence homology, stimulate ELL elongation activity (7Kong S.E. Banks C.A. Shilatifard A. Conaway J.W. Conaway R.C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10094-10098Crossref PubMed Scopus (69) Google Scholar). Studies by Luo et al. (8Luo R.T. Lavau C. Du C. Simone F. Polak P.E. Kawamata S. Thirman M.J. Mol. Cell. Biol. 2001; 21: 5678-5687Crossref PubMed Scopus (70) Google Scholar) argued that EAF proteins are important in MLL-ELL leukemogenesis, whereas our previous studies showed that EAF2/U19 inhibits xenograft prostate tumor growth and undergoes down-regulation in prostate cancer cell lines (9Xiao W. Zhang Q. Jiang F. Pins M. Kozlowski J.M. Wang Z. Cancer Res. 2003; 63: 4698-4704PubMed Google Scholar). These findings link the EAF2/U19 gene with two major human cancers: prostate cancer and acute myeloid leukemia. To investigate the function EAF2/U19 in vivo, we constructed a murine knock-out model. The EAF2/U19 knock-out mice develop B-cell lymphoma, lung adenocarcinoma, hepatocellular carcinoma, and prostate intraepithelial neoplasia with high frequency (10Xiao W. Zhang Q. Habermacher G. Yang X. Zhang A.Y. Cai X. Hahn J. Liu J. Pins M. Doglio L. Dhir R. Gingrich J. Wang Z. Oncogene. 2008; 27: 1536-1544Crossref PubMed Scopus (52) Google Scholar). In addition, we demonstrated that EAF2/U19 could bind to and stabilize the classic tumor suppressor-pVHL (11Xiao W. Ai J. Habermacher G. Volpert O. Yang X. Zhang A.Y. Hahn J. Cai X. Wang Z. Cancer Res. 2009; 69: 2599-2606Crossref PubMed Scopus (26) Google Scholar). These findings further support EAF2/U19 as a potential tumor suppressor; however, the molecular mechanisms underlying this activity remain poorly defined. Evidence suggests that oncogenes and tumor suppressors may play a role in normal vertebrate embryogenesis. Thus, the evaluation of how these proteins function during embryogenesis would not only lead to a better understanding of development but may also shed light on how these proteins contribute to tumor initiation and progression (12Lee H. Kimelman D. Dev. Cell. 2002; 2: 607-616Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 13Langheinrich U. Hennen E. Stott G. Vacun G. Curr. Biol. 2002; 12: 2023-2028Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Several studies have shown that eaf2/u19 appears to be important during embryogenesis, particularly in eye development (14Li M. Wu X. Zhuang F. Jiang S. Jiang M. Liu Y.H. Dev. Dyn. 2003; 228: 273-280Crossref PubMed Scopus (19) Google Scholar, 15Maurus D. Héligon C. Bürger-Schwärzler A. Brändli A.W. Kühl M. EMBO J. 2005; 24: 1181-1191Crossref PubMed Scopus (86) Google Scholar), but its function in vertebrate embryogenesis remains unclear. In vertebrates, the body plan is established during gastrulation by a set of morphogenetic processes, including epiboly, internalization, and convergence and extension movements (16Myers D.C. Sepich D.S. Solnica-Krezel L. Trends Genet. 2002; 18: 447-455Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Gastrulation starts with the epiboly of the enveloping layer and deep cells and the internalization of prospective mesendodermal cells, followed by convergence and extension movements. convergence and extension movements narrow embryonic tissues mediolaterally and lengthen them anteroposteriorly (17Keller R. Shih J. Domingo C. Dev. Suppl. 1992; : 81-91PubMed Google Scholar). By convergence movements, the precursor cells of many organs migrate toward midline from bilateral tissues and assemble to form the primitive organs (18Stainier D.Y. Nat. Rev. 2001; 2: 39-48Crossref Scopus (321) Google Scholar, 19Ober E.A. Olofsson B. Mäkinen T. Jin S.W. Shoji W. Koh G.Y. Alitalo K. Stainier D.Y. EMBO Rep. 2004; 5: 78-84Crossref PubMed Scopus (86) Google Scholar). During the convergence and extension movements, multiple signaling cascades control coordinated cell behaviors. silberblick/wnt11 (slb) and pipetail/wnt5 (ppt) zebrafish mutants show abnormal convergence and extension movements and display a short tail and body axis (20Heisenberg C.P. Tada M. Rauch G.J. Saúde L. Concha M.L. Geisler R. Stemple D.L. Smith J.C. Wilson S.W. Nature. 2000; 405: 76-81Crossref PubMed Scopus (826) Google Scholar, 21Kilian B. Mansukoski H. Barbosa F.C. Ulrich F. Tada M. Heisenberg C.P. Mech. Dev. 2003; 120: 467-476Crossref PubMed Scopus (258) Google Scholar). Thus, the noncanonical Wnt cascade has been proposed to be essential for convergence and extension movements. In addition, rhoA has been reported to act downstream of wnt5 and wnt11 to regulate convergence and extension movements (22Zhu S. Liu L. Korzh V. Gong Z. Low B.C. Cell. Signal. 2006; 18: 359-372Crossref PubMed Scopus (92) Google Scholar). We employed the zebrafish embryo as a model system to investigate the role of eaf1 and eaf2/u19 in vertebrate development. Based on initial findings, we focused our examination on the regulation of convergence and extension movements and midline convergence of the heart and pancreas primordia by Eaf1 and Eaf2/U19 during zebrafish embryogenesis. We also examined whether eaf1 and eaf2/u19 regulate convergence and extension movements by noncanonical Wnt signaling. DISCUSSIONPrevious studies have established that EAF2/U19 contributes to tumor suppression (9Xiao W. Zhang Q. Jiang F. Pins M. Kozlowski J.M. Wang Z. Cancer Res. 2003; 63: 4698-4704PubMed Google Scholar, 10Xiao W. Zhang Q. Habermacher G. Yang X. Zhang A.Y. Cai X. Hahn J. Liu J. Pins M. Doglio L. Dhir R. Gingrich J. Wang Z. Oncogene. 2008; 27: 1536-1544Crossref PubMed Scopus (52) Google Scholar). Other studies suggest that EAF1 and EAF2/U19, like other tumor suppressors, may play a role in vertebrate development (14Li M. Wu X. Zhuang F. Jiang S. Jiang M. Liu Y.H. Dev. Dyn. 2003; 228: 273-280Crossref PubMed Scopus (19) Google Scholar, 15Maurus D. Héligon C. Bürger-Schwärzler A. Brändli A.W. Kühl M. EMBO J. 2005; 24: 1181-1191Crossref PubMed Scopus (86) Google Scholar). However, their exact function in embryogenesis remains unclear. To better understand the cellular function of EAF1 and EAF2/U19 and subsequently how these proteins suppress tumors will require a more extensive investigation of their roles in embryogenesis. Here, we are the first to present data showing that eaf1 and eaf2/u19 mediate effective convergence and extension movements through the maintenance of wnt11 and wnt5 expression during zebrafish gastrulation.Eaf family members demonstrated a high degree of conservation across species and with each other, so not surprisingly, the expression of eaf1 and eaf2/u19 observed in the zebrafish mirrored the pattern seen in the mouse (14Li M. Wu X. Zhuang F. Jiang S. Jiang M. Liu Y.H. Dev. Dyn. 2003; 228: 273-280Crossref PubMed Scopus (19) Google Scholar). However, intercrosses of EAF2/U19 heterozygous knock-out mice yielded Eaf2/U19-null offspring at Mendelian ratios at birth, suggesting that early mouse development did not require EAF2/U19 (10Xiao W. Zhang Q. Habermacher G. Yang X. Zhang A.Y. Cai X. Hahn J. Liu J. Pins M. Doglio L. Dhir R. Gingrich J. Wang Z. Oncogene. 2008; 27: 1536-1544Crossref PubMed Scopus (52) Google Scholar). Eaf2/U19 knockdown in zebrafish embryos alone caused defects in convergence and extension movements in our study. This phenotypic difference between mouse and zebrafish might result from either the redundant function of EAF1 and EAF2/U19 in mouse embryogenesis or from slightly different functions of EAF2/U19 in mammalians and lower vertebrate fish. Indeed, functional differences between mammalian and fish genes are not uncommon (36Correa R.G. Matsui T. Tergaonkar V. Rodriguez-Esteban C. Izpisua-Belmonte J.C. Verma I.M. Curr. Biol. 2005; 15: 1291-1295Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In addition, Eaf2/U19 knockdown in Xenopus laevis caused defects in eye development, which was a pure eye phenotype unrelated to the defect of convergence and extension movement (15Maurus D. Héligon C. Bürger-Schwärzler A. Brändli A.W. Kühl M. EMBO J. 2005; 24: 1181-1191Crossref PubMed Scopus (86) Google Scholar). However, eaf2/u19 knockdown in zebrafish caused shorter eye distance or eye fusion (partial cyclopia) resulting from the defect in convergence and extension movements (Fig. 8C). Together, eaf1 and eaf2/u19 might play different roles in eye development during embryogenesis. Further demonstrating the different mechanism of eaf1 and eaf2/u19 playing their roles in eye formation among species probably will give us a more complete picture about the function of eaf1 and eaf2/u19 in embryogenesis.eaf1 and eaf2/u19 appeared to play partially redundant roles in the regulation of convergence and extension movements. Knockdown of both eaf1 and eaf2/u19 resulted in abnormal cell behavior in both the head and trunk mesendoderm (Fig. 2). The phenotypes observed in eafs morphants included disorganized head structure, reduced body axis, and shorter tail and were similar to those seen in embryos with a disruption of convergence and extension movements (20Heisenberg C.P. Tada M. Rauch G.J. Saúde L. Concha M.L. Geisler R. Stemple D.L. Smith J.C. Wilson S.W. Nature. 2000; 405: 76-81Crossref PubMed Scopus (826) Google Scholar, 21Kilian B. Mansukoski H. Barbosa F.C. Ulrich F. Tada M. Heisenberg C.P. Mech. Dev. 2003; 120: 467-476Crossref PubMed Scopus (258) Google Scholar, 33Wallingford J.B. Fraser S.E. Harland R.M. Dev. Cell. 2002; 2: 695-706Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 37Yamashita S. Miyagi C. Carmany-Rampey A. Shimizu T. Fujii R. Schier A.F. Hirano T. Dev. Cell. 2002; 2: 363-375Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 38Topczewski J. Sepich D.S. Myers D.C. Walker C. Amores A. Lele Z. Hammerschmidt M. Postlethwait J. Solnica-Krezel L. Dev. Cell. 2001; 1: 251-264Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 39Jessen J.R. Topczewski J. Bingham S. Sepich D.S. Marlow F. Chandrasekhar A. Solnica-Krezel L. Nat. Cell Biol. 2002; 4: 610-615Crossref PubMed Scopus (396) Google Scholar). On the basis of phenotype, the marker gene expression patterns, and cell tracing experiments, we concluded that eaf1 and eaf2/u19 served as novel regulators of convergence and extension movements (FIGURE 2, FIGURE 3, FIGURE 4). In reciprocal rescue experiments, eaf1 mRNA could rescue Eaf2-MO1 knockdowns, and eaf2/u19 mRNA could rescue Eaf1-MO knockdowns, suggesting redundant roles for Eaf1 and Eaf2/U19 in the regulation of convergence and extension movements. However, knockdown of either eaf1 or eaf2/u19 produced similar defects that increased in severity with the combined knockdown of both Eaf1 and Eaf2/U19 (FIGURE 2, FIGURE 3, FIGURE 4), indicating that these proteins do not have completely redundant functions.In this study, we found that eaf1 and eaf2/u19 might contribute to the regulation of convergence and extension movements through noncanonical Wnt signaling. Eaf1 and Eaf2/U19 knockdown dramatically down-regulated wnt11 expression, whereas introduction of ectopic eaf1 and eaf2/u19 mRNA increased wnt11 expression (Fig. 7). Moreover, wnt11 mRNA rescued convergence and extension movement defects caused by Eaf1 and Eaf2/U19 knockdown with high frequency (Fig. 9A). However, overexpression of wnt11 by mRNA injection did not affect the expression of eaf1 and eaf2/u19 (Fig. 8). These findings imply that eaf1 and eaf2/u19 act upstream of wnt11 to control its expression and to govern convergence and extension movements. Furthermore, Eaf1 and Eaf2/U19 knockdown down-regulated wnt5 expression (Fig. 7, A and C), and wnt5 mRNA, in turn, also rescued defects in convergence and extension movements caused by the loss of eaf1 and eaf2/u19 (Fig. 9A (A4)). However, overexpression of eaf1 and eaf2/u19 also down-regulated wnt5 expression, with expression levels comparable with those seen in the eafs morphants (Fig. 7, E and G). This suggests that the maintenance of wnt5 might require a specific level or ratio of eaf1 and eaf2/u19. Similar to that of wnt11, wnt5 overexpression did not affect the expression of eaf1 and eaf2/u19, suggesting that eaf1 and eaf2/u19 also act upstream of wnt5. As a downstream activator of noncanonical Wnt ligands, RhoA rescued convergence and extension movement defect more effectively than Wnt5 and Wnt11 (Fig. 8), indicating a convergence of wnt5 and wnt11 on rhoA during the regulation of convergence and extension movements. Of note, the expression patterns of eaf1 and eaf2/u19 were similar to that of rhoA during zebrafish and Xenopus embryogenesis (22Zhu S. Liu L. Korzh V. Gong Z. Low B.C. Cell. Signal. 2006; 18: 359-372Crossref PubMed Scopus (92) Google Scholar, 40Wünnenberg-Stapleton K. Blitz I.L. Hashimoto C. Cho K.W. Development. 1999; 126: 5339-5351Crossref PubMed Google Scholar).Notably, eaf1 and eaf2/u19 have activity in stimulating ELL transcriptional elongation (7Kong S.E. Banks C.A. Shilatifard A. Conaway J.W. Conaway R.C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10094-10098Crossref PubMed Scopus (69) Google Scholar). Thus, if knockdown of eaf1 and eaf2/u19 only caused inhibition of gene expression, eafs regulating the expression of Wnts (wnt11, wnt5, and wnt11r) presented in this study might be nonspecific as a result of disrupting the general function of eafs as regulators of transcriptional elongation. However, in addition to wnt4a,4 foxo5 (Fig. 9A) as well as other genes5 were up-regulated by eafs knockdown, unrelated to their function as regulators of transcriptional elongation. Therefore, eafs knockdown inhibiting the expression of Wnts appears to be specific. Taking into consideration that the knockdown of eafs did not affect cell fate specification at the beginning of gastrulation, it suggests that eafs might directly regulate noncanonical Wnt ligands (especially for wnt11). However, this conclusion needs to be further verified by more direct ways, such as promoter chromatin immunoprecipitation assays, etc.Knockdown of Eaf1 and Eaf2/U19 protein levels also resulted in the failure of fusion of the heart and pancreas primordia (Fig. 6), but neither endoderm fate determination genes nor myocardial precursor marker genes displayed significant changes in their expression levels (Fig. 6, F–H). These observations imply that eaf1 and eaf2/u19 act as novel regulators of midline convergence of both endoderm- and mesoderm-derived organ primordia without affecting the specification of progenitors.Although a primary role of non-canonical Wnt signaling was to govern convergence and extension movements, studies had also shown that the three Wnt noncanonical ligands, wnt4a, wnt11, and wnt11r, redundantly regulated midline convergence of organ primordia, in zebrafish embryos (31Matsui T. Raya A. Kawakami Y. Callol-Massot C. Capdevila J. Rodríguez-Esteban C. Izpisúa Belmonte J.C. Genes Dev. 2005; 19: 164-175Crossref PubMed Scopus (133) Google Scholar, 41Miyasaka K.Y. Kida Y.S. Sato T. Minami M. Ogura T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11274-11279Crossref PubMed Scopus (33) Google Scholar). In this study, we showed that the midline migration of heart precursors and pancreas precursors required expression of eaf1 and eaf2/u19 (Fig. 6). We also showed that eaf1 and eaf2/u19 mediated midline convergence through noncanonical Wnt signaling, specifically by regulating expression of wnt11 and wnt11r (). Unexpectedly, however, knockdown of eaf1 and eaf2/u19 up-regulated wnt4a expression.4 This observation suggested that, unlike wnt11 and wntllr, wnt4a was not a downstream factor of eaf1 and eaf2/u19 in the signaling pathway that regulated midline convergence of organ precursors.In conclusion, our data are the first to demonstrate that eaf1 and eaf2/u19 have essential roles in regulating embryonic cell behavior and migration. Although the complete pathway has yet to be defined, our study shows that eaf1 and eaf2/u19 function in this pathway by specifically modulating expression of wnt11 and wnt5, which in turn converge on rhoA for the positive regulation of convergence and extension movements. Previous studies have established that EAF2/U19 contributes to tumor suppression (9Xiao W. Zhang Q. Jiang F. Pins M. Kozlowski J.M. Wang Z. Cancer Res. 2003; 63: 4698-4704PubMed Google Scholar, 10Xiao W. Zhang Q. Habermacher G. Yang X. Zhang A.Y. Cai X. Hahn J. Liu J. Pins M. Doglio L. Dhir R. Gingrich J. Wang Z. Oncogene. 2008; 27: 1536-1544Crossref PubMed Scopus (52) Google Scholar). Other studies suggest that EAF1 and EAF2/U19, like other tumor suppressors, may play a role in vertebrate development (14Li M. Wu X. Zhuang F. Jiang S. Jiang M. Liu Y.H. Dev. Dyn. 2003; 228: 273-280Crossref PubMed Scopus (19) Google Scholar, 15Maurus D. Héligon C. Bürger-Schwärzler A. Brändli A.W. Kühl M. EMBO J. 2005; 24: 1181-1191Crossref PubMed Scopus (86) Google Scholar). However, their exact function in embryogenesis remains unclear. To better understand the cellular function of EAF1 and EAF2/U19 and subsequently how these proteins suppress tumors will require a more extensive investigation of their roles in embryogenesis. Here, we are the first to present data showing that eaf1 and eaf2/u19 mediate effective convergence and extension movements through the maintenance of wnt11 and wnt5 expression during zebrafish gastrulation. Eaf family members demonstrated a high degree of conservation across species and with each other, so not surprisingly, the expression of eaf1 and eaf2/u19 observed in the zebrafish mirrored the pattern seen in the mouse (14Li M. Wu X. Zhuang F. Jiang S. Jiang M. Liu Y.H. Dev. Dyn. 2003; 228: 273-280Crossref PubMed Scopus (19) Google Scholar). However, intercrosses of EAF2/U19 heterozygous knock-out mice yielded Eaf2/U19-null offspring at Mendelian ratios at birth, suggesting that early mouse development did not require EAF2/U19 (10Xiao W. Zhang Q. Habermacher G. Yang X. Zhang A.Y. Cai X. Hahn J. Liu J. Pins M. Doglio L. Dhir R. Gingrich J. Wang Z. Oncogene. 2008; 27: 1536-1544Crossref PubMed Scopus (52) Google Scholar). Eaf2/U19 knockdown in zebrafish embryos alone caused defects in convergence and extension movements in our study. This phenotypic difference between mouse and zebrafish might result from either the redundant function of EAF1 and EAF2/U19 in mouse embryogenesis or from slightly different functions of EAF2/U19 in mammalians and lower vertebrate fish. Indeed, functional differences between mammalian and fish genes are not uncommon (36Correa R.G. Matsui T. Tergaonkar V. Rodriguez-Esteban C. Izpisua-Belmonte J.C. Verma I.M. Curr. Biol. 2005; 15: 1291-1295Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In addition, Eaf2/U19 knockdown in Xenopus laevis caused defects in eye development, which was a pure eye phenotype unrelated to the defect of convergence and extension movement (15Maurus D. Héligon C. Bürger-Schwärzler A. Brändli A.W. Kühl M. EMBO J. 2005; 24: 1181-1191Crossref PubMed Scopus (86) Google Scholar). However, eaf2/u19 knockdown in zebrafish caused shorter eye distance or eye fusion (partial cyclopia) resulting from the defect in convergence and extension movements (Fig. 8C). Together, eaf1 and eaf2/u19 might play different roles in eye development during embryogenesis. Further demonstrating the different mechanism of eaf1 and eaf2/u19 playing their roles in eye formation among species probably will give us a more complete picture about the function of eaf1 and eaf2/u19 in embryogenesis. eaf1 and eaf2/u19 appeared to play partially redundant roles in the regulation of convergence and extension movements. Knockdown of both eaf1 and eaf2/u19 resulted in abnormal cell behavior in both the head and trunk mesendoderm (Fig. 2). The phenotypes observed in eafs morphants included disorganized head structure, reduced body axis, and shorter tail and were similar to those seen in embryos with a disruption of convergence and extension movements (20Heisenberg C.P. Tada M. Rauch G.J. Saúde L. Concha M.L. Geisler R. Stemple D.L. Smith J.C. Wilson S.W. Nature. 2000; 405: 76-81Crossref PubMed Scopus (826) Google Scholar, 21Kilian B. Mansukoski H. Barbosa F.C. Ulrich F. Tada M. Heisenberg C.P. Mech. Dev. 2003; 120: 467-476Crossref PubMed Scopus (258) Google Scholar, 33Wallingford J.B. Fraser S.E. Harland R.M. Dev. Cell. 2002; 2: 695-706Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 37Yamashita S. Miyagi C. Carmany-Rampey A. Shimizu T. Fujii R. Schier A.F. Hirano T. Dev. Cell. 2002; 2: 363-375Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 38Topczewski J. Sepich D.S. Myers D.C. Walker C. Amores A. Lele Z. Hammerschmidt M. Postlethwait J. Solnica-Krezel L. Dev. Cell. 2001; 1: 251-264Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 39Jessen J.R. Topczewski J. Bingham S. Sepich D.S. Marlow F. Chandrasekhar A. Solnica-Krezel L. Nat. Cell Biol. 2002; 4: 610-615Crossref PubMed Scopus (396) Google Scholar). On the basis of phenotype, the marker gene expression patterns, and cell tracing experiments, we concluded that eaf1 and eaf2/u19 served as novel regulators of convergence and extension movements (FIGURE 2, FIGURE 3, FIGURE 4). In reciprocal rescue experiments, eaf1 mRNA could rescue Eaf2-MO1 knockdowns, and eaf2/u19 mRNA could rescue Eaf1-MO knockdowns, suggesting redundant roles for Eaf1 and Eaf2/U19 in the regulation of convergence and extension movements. However, knockdown of either eaf1 or eaf2/u19 produced similar defects that increased in severity with the combined knockdown of both Eaf1 and Eaf2/U19 (FIGURE 2, FIGURE 3, FIGURE 4), indicating that these proteins do not have completely redundant functions. In this study, we found that eaf1 and eaf2/u19 might contribute to the regulation of convergence and extension movements through noncanonical Wnt signaling. Eaf1 and Eaf2/U19 knockdown dramatically down-regulated wnt11 expression, whereas introduction of ectopic eaf1 and eaf2/u19 mRNA increased wnt11 expression (Fig. 7). Moreover, wnt11 mRNA rescued convergence and extension movement defects caused by Eaf1 and Eaf2/U19 knockdown with high frequency (Fig. 9A). However, overexpression of wnt11 by mRNA injection did not affect the expression of eaf1 and eaf2/u19 (Fig. 8). These findings imply that eaf1 and eaf2/u19 act upstream of wnt11 to control its expression and to govern convergence and extension movements. Furthermore, Eaf1 and Eaf2/U19 knockdown down-regulated wnt5 expression (Fig. 7, A and C), and wnt5 mRNA, in turn, also rescued defects in convergence and extension movements caused by the loss of eaf1 and eaf2/u19 (Fig. 9A (A4)). However, overexpression of eaf1 and eaf2/u19 also down-regulated wnt5 expression, with expression levels comparable with those seen in the eafs morphants (Fig. 7, E and G). This suggests that the maintenance of wnt5 might require a specific level or ratio of eaf1 and eaf2/u19. Similar to that of wnt11, wnt5 overexpression did not affect the expression of eaf1 and eaf2/u19, suggesting that eaf1 and eaf2/u19 also act upstream of wnt5. As a downstream activator of noncanonical Wnt ligands, RhoA rescued convergence and extension movement defect more effectively than Wnt5 and Wnt11 (Fig. 8), indicating a convergence of wnt5 and wnt11 on rhoA during the regulation of convergence and extension movements. Of note, the expression patterns of eaf1 and eaf2/u19 were similar to that of rhoA during zebrafish and Xenopus embryogenesis (22Zhu S. Liu L. Korzh V. Gong Z. Low B.C. Cell. Signal. 2006; 18: 359-372Crossref PubMed Scopus (92) Google Scholar, 40Wünnenberg-Stapleton K. Blitz I.L. Hashimoto C. Cho K.W. Development. 1999; 126: 5339-5351Crossref PubMed Google Scholar). Notably, eaf1 and eaf2/u19 have activity in stimulating ELL transcriptional elongation (7Kong S.E. Banks C.A. Shilatifard A. Conaway J.W. Conaway R.C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10094-10098Crossref PubMed Scopus (69) Google Scholar). Thus, if knockdown of eaf1 and eaf2/u19 only caused inhibition of gene expression, eafs regulating the expression of Wnts (wnt11, wnt5, and wnt11r) presented in this study might be nonspecific as a result of disrupting the general function of eafs as regulators of transcriptional elongation. However, in addition to wnt4a,4 foxo5 (Fig. 9A) as well as other genes5 were up-regulated by eafs knockdown, unrelated to their function as regulators of transcriptional elongation. Therefore, eafs knockdown inhibiting the expression of Wnts appears to be specific. Taking into consideration that the knockdown of eafs did not affect cell fate specification at the beginning of gastrulation, it suggests that eafs might directly regulate noncanonical Wnt ligands (especially for wnt11). However, this conclusion needs to be further verified by more direct ways, such as promoter chromatin immunoprecipitation assays, etc. Knockdown of Eaf1 and Eaf2/U19 protein levels also resulted in the failure of fusion of the heart and pancreas primordia (Fig. 6), but neither endoderm fate determination genes nor myocardial precursor marker genes displayed significant changes in their expression levels (Fig. 6, F–H). These observations imply that eaf1 and eaf2/u19 act as novel regulators of midline convergence of both endoderm- and mesoderm-derived organ primordia without affecting the specification of progenitors. Although a primary role of non-canonical Wnt signaling was to govern convergence and extension movements, studies had also shown that the three Wnt noncanonical ligands, wnt4a, wnt11, and wnt11r, redundantly regulated midline convergence of organ primordia, in zebrafish embryos (31Matsui T. Raya A. Kawakami Y. Callol-Massot C. Capdevila J. Rodríguez-Esteban C. Izpisúa Belmonte J.C. Genes Dev. 2005; 19: 164-175Crossref PubMed Scopus (133) Google Scholar, 41Miyasaka K.Y. Kida Y.S. Sato T. Minami M. Ogura T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11274-11279Crossref PubMed Scopus (33) Google Scholar). In this study, we showed that the midline migration of heart precursors and pancreas precursors required expression of eaf1 and eaf2/u19 (Fig. 6). We also showed that eaf1 and eaf2/u19 mediated midline convergence through noncanonical Wnt signaling, specifically by regulating expression of wnt11 and wnt11r (). Unexpectedly, however, knockdown of eaf1 and eaf2/u19 up-regulated wnt4a expression.4 This observation suggested that, unlike wnt11 and wntllr, wnt4a was not a downstream factor of eaf1 and eaf2/u19 in the signaling pathway that regulated midline convergence of organ precursors. In conclusion, our data are the first to demonstrate that eaf1 and eaf2/u19 have essential roles in regulating embryonic cell behavior and migration. Although the complete pathway has yet to be defined, our study shows that eaf1 and eaf2/u19 function in this pathway by specifically modulating expression of wnt11 and wnt5, which in turn converge on rhoA for the positive regulation of convergence and extension movements. We thank Drs. Zhan Yin, Yonghua Sun, and T. Whitfield for the generous gifts of various reagents. We also thank Moira Hitchens for editing." @default.
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• W2088903662 title "Zebrafish eaf1 and eaf2/u19 Mediate Effective Convergence and Extension Movements through the Maintenance of wnt11 and wnt5 Expression" @default.
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