title: Epithelial cell fate in the nephron tubule is mediated by the ETS transcription factors etv5a and etv4 during zebrafish kidney development.
abstract: Kidney development requires the differentiation and organization of discrete nephron epithelial lineages, yet the genetic and molecular pathways involved in these events remain poorly understood. The embryonic zebrafish kidney, or pronephros, provides a simple and useful model to study nephrogenesis. The pronephros is primarily comprised of two types of epithelial cells: transportive and multiciliated cells (MCCs). Transportive cells occupy distinct tubule segments and are characterized by the expression of various solute transporters, while MCCs function in fluid propulsion and are dispersed in a "salt-and-pepper" fashion within the tubule. Epithelial cell identity is reliant on interplay between the Notch signaling pathway and retinoic acid (RA) signaling, where RA promotes MCC fate by inhibiting Notch activity in renal progenitors, while Notch acts downstream to trigger transportive cell formation and block adoption of an MCC identity. Previous research has shown that the transcription factor ets variant 5a (etv5a), and its closely related ETS family members, are required for ciliogenesis in other zebrafish tissues. Here, we mapped etv5a expression to renal progenitors that occupy domains where MCCs later emerge. Thus, we hypothesized that etv5a is required for normal development of MCCs in the nephron. etv5a loss of function caused a decline of MCC number as indicated by the reduced frequency of cells that expressed the MCC-specific markers outer dense fiber of sperm tails 3b (odf3b) and centrin 4 (cetn4), where rescue experiments partially restored MCC incidence. Interestingly, deficiency of ets variant 4 (etv4), a related gene that is broadly expressed in the posterior mesoderm during somitogenesis stages, also led to reduced MCC numbers, which were further reduced by dual etv5a/4 deficiency, suggesting that both of these ETS factors are essential for MCC formation and that they also might have redundant activities. In epistatic studies, exogenous RA treatment expanded the etv5a domain within the renal progenitor field and RA inhibition blocked etv5a in this populace, indicating that etv5a acts downstream of RA. Additionally, treatment with exogenous RA partially rescued the reduced MCC phenotype after loss of etv5a. Further, abrogation of Notch with the small molecule inhibitor DAPT increased the renal progenitor etv5a expression domain as well as MCC density in etv5a deficient embryos, suggesting Notch acts upstream to inhibit etv5a. In contrast, etv4 levels in renal progenitors were unaffected by changes in RA or Notch signaling levels, suggesting a possible non-cell autonomous role during pronephros formation. Taken together, these findings have revealed new insights about the genetic mechanisms of epithelial cell development during nephrogenesis.
authors.affiliation: Department of Biological Sciences, Center for Zebrafish Research, Center for Stem Cells and Regenerative Medicine, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Amanda N
authors.initials: AN
authors.lastname: Marra
authors.affiliation: Department of Biological Sciences, Center for Zebrafish Research, Center for Stem Cells and Regenerative Medicine, University of Notre Dame, Notre Dame, IN 46556, USA. Electronic address: rwingert@nd.edu.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Wingert
mesh_terms: D000165:Acridine Orange / Q000737:chemistry; D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D004847:Epithelial Cells / Q000166:cytology*; D018507:Gene Expression Regulation, Developmental; D017403:In Situ Hybridization; D017404:In Situ Hybridization, Fluorescence; D007668:Kidney / Q000196:embryology*; D009399:Nephrons / Q000196:embryology*; D038081:Organogenesis / Q000235:genetics; D060910:Pronephros / Q000196:embryology*; D050783:Proto-Oncogene Proteins c-ets / Q000502:physiology*; D051880:Receptors, Notch / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000502:physiology*; D014212:Tretinoin / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology* / Q000502:physiology; D029961:Zebrafish Proteins / Q000502:physiology*
chemical_list: C000590222:Etv5a protein, zebrafish; D050783:Proto-Oncogene Proteins c-ets; D051880:Receptors, Notch; D014157:Transcription Factors; D029961:Zebrafish Proteins; C065788:transcription factor PEA3; D014212:Tretinoin; D000165:Acridine Orange
keywords: Epithelial cell fate; Kidney; Multiciliated cells; Nephrogenesis; Notch signaling; Pronephros; Retinoic acid; etv4; etv5a
references.citation: Mech Dev. 1999 Nov;88(2):233-6
references.pmid: 10534622
references.citation: Exp Hematol. 2008 Apr;36(4):424-32
references.pmid: 18243489
references.citation: Cells. 2015 Sep 11;4(3):483-99
references.pmid: 26378582
references.citation: Curr Biol. 2014 Oct 6;24(19):R973-82
references.pmid: 25291643
references.citation: Dev Biol. 1997 Aug 15;188(2):189-204
references.pmid: 9268568
references.citation: Development. 2010 Jun;137(12):1975-9
references.pmid: 20463033
references.citation: Dev Dyn. 2007 Nov;236(11):3100-10
references.pmid: 17907201
references.citation: Genesis. 2012 Mar;50(3):176-85
references.pmid: 22083727
references.citation: Cell Dev Biol. 2014 Jan 1;3(1):1000131
references.pmid: 24855634
references.citation: J Endocrinol. 2008 Aug;198(2):281-90
references.pmid: 18492810
references.citation: Birth Defects Res C Embryo Today. 2014 Dec;102(4):374-81
references.pmid: 25420794
references.citation: J Vis Exp. 2009;(25). pii: 1229. doi: 10.3791/1229
references.pmid: 19322135
references.citation: Trends Biochem Sci. 1998 Jun;23(6):213-6
references.pmid: 9644975
references.citation: PLoS Genet. 2007 Oct;3(10):1922-38
references.pmid: 17953490
references.citation: Methods Cell Biol. 2010;97:415-35
references.pmid: 20719283
references.citation: J Biol Chem. 2015 Sep 11;290(37):22460-73
references.pmid: 26224636
references.citation: Dev Biol. 2010 Jun 1;342(1):11-25
references.pmid: 20346941
references.citation: Kidney Int. 2008 May;73(10):1120-7
references.pmid: 18322540
references.citation: Gene Expr Patterns. 2006 Oct;6(8):777-82
references.pmid: 16516559
references.citation: Dev Dyn. 1995 Jul;203(3):253-310
references.pmid: 8589427
references.citation: Biochim Biophys Acta. 1998 Apr 17;1377(2):F1-11
references.pmid: 9606973
references.citation: Mech Dev. 2001 Sep;107(1-2):105-17
references.pmid: 11520667
references.citation: Dev Biol. 2014 Feb 1;386(1):111-22
references.pmid: 24309209
references.citation: Exp Hematol. 2008 Sep;36(9):1132-42
references.pmid: 18550261
references.citation: Nature. 2005 Aug 18;436(7053):1035-39
references.pmid: 16110529
references.citation: J Vis Exp. 2014;(89). doi: 10.3791/51604
references.pmid: 25078510
references.citation: J Am Soc Nephrol. 2013 Jan;24(1):77-87
references.pmid: 23160512
references.citation: Am J Respir Crit Care Med. 1996 Dec;154(6 Pt 1):1868-902
references.pmid: 8970383
references.citation: Dev Biol. 2014 Dec 15;396(2):183-200
references.pmid: 25446529
references.citation: Development. 2005 Apr;132(8):1907-21
references.pmid: 15790966
references.citation: Development. 2013 Jul;140(13):2755-64
references.pmid: 23698347
references.citation: Blood. 2009 Mar 19;113(12):2843-50
references.pmid: 19047682
references.citation: Am J Kidney Dis. 2015 Jul;66(1 Suppl 1):Svii, S1-305
references.pmid: 26111994
references.citation: Development. 2007 Mar;134(6):1111-22
references.pmid: 17287248
references.citation: Elife. 2015;4. doi: 10.7554/eLife.07405
references.pmid: 26057828
references.citation: Curr Biol. 2001 Apr 3;11(7):503-7
references.pmid: 11413000
references.citation: Science. 1963 Jan 18;139(3551):221-2
references.pmid: 14001896
references.citation: Am J Kidney Dis. 2005 Jun;45(6):1096-9
references.pmid: 15957140
references.citation: J Cell Sci. 2013 Dec 15;126(Pt 24):5626-34
references.pmid: 24101720
references.citation: Dev Biol. 2015 Mar 1;399(1):100-16
references.pmid: 25542995
references.citation: Annu Rev Cell Dev Biol. 2006;22:509-29
references.pmid: 16822174
references.citation: PLoS Genet. 2007 Jan 26;3(1):e18
references.pmid: 17257056
references.citation: Dev Dyn. 2011 Aug;240(8):2011-27
references.pmid: 21761484
references.citation: J Am Soc Nephrol. 2005 Feb;16(2):299-304
references.pmid: 15647335
references.citation: Gene Expr Patterns. 2014 Nov;16(2):104-13
references.pmid: 25460834
references.citation: Nat Genet. 2009 Dec;41(12):1295-302
references.pmid: 19898483
references.citation: Curr Opin Nephrol Hypertens. 2011 Jul;20(4):416-24
references.pmid: 21519251
references.citation: Am J Pathol. 1968 Oct;53(4):609-16
references.pmid: 5677142
references.citation: Ultrastruct Pathol. 1984;6(4):285-94
references.pmid: 6485121
references.citation: Wiley Interdiscip Rev Dev Biol. 2013 Sep-Oct;2(5):559-85
references.pmid: 24014448
references.citation: Dev Cell. 2009 Apr;16(4):600-6
references.pmid: 19386268
references.citation: Transl Res. 2014 Feb;163(2):109-22
references.pmid: 24183931
references.citation: Development. 2004 Dec;131(24):6225-35
references.pmid: 15563524
references.citation: Ultrastruct Pathol. 1995 May-Jun;19(3):201-3
references.pmid: 7631434
references.citation: J Cell Sci. 2006 May 15;119(Pt 10):2127-37
references.pmid: 16638803
references.citation: Cells. 2015 May 27;4(2):218-33
references.pmid: 26024215
references.citation: J Exp Zool Suppl. 1989;2:186-92
references.pmid: 2575649
references.citation: Organogenesis. 2010 Oct-Dec;6(4):252-62
references.pmid: 21220964
references.citation: Dev Cell. 2010 May 18;18(5):698-712
references.pmid: 20493806
references.citation: Dev Biol. 2011 Jun 1;354(1):134-42
references.pmid: 21466798
references.citation: Genome Res. 2001 Dec;11(12):1979-87
references.pmid: 11731487
references.citation: Beitr Pathol. 1974 Mar;151(3):297-303
references.pmid: 4857844
references.citation: Dev Biol. 2011 Oct 15;358(2):318-30
references.pmid: 21871448
references.citation: Development. 2013 Aug;140(15):3095-106
references.pmid: 23824578
references.citation: Beitr Pathol. 1971 Jun;143(3):227-40
references.pmid: 5559019
references.citation: Biochim Biophys Acta. 2012 Aug;1826(1):1-12
references.pmid: 22425584
references.citation: Cold Spring Harb Perspect Biol. 2012 May;4(5). pii: a008300. doi: 10.1101/cshperspect.a008300
references.pmid: 22550230
references.citation: Oncogene. 1998 Jul 9;17(1):93-104
references.pmid: 9671318