Pax8 and Pax2 are specifically required at different steps of Xenopus pronephros development

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1 Pax8 and Pax2 are specifically required at different steps of Xenopus pronephros development Isabelle Buisson, Ronan Le Bouffant, Mélinée Futel, Jean-François Riou, Muriel Umbhauer To cite this version: Isabelle Buisson, Ronan Le Bouffant, Mélinée Futel, Jean-François Riou, Muriel Umbhauer. Pax8 and Pax2 are specifically required at different steps of Xenopus pronephros development. Developmental Biology, Elsevier, 2014, 397 (2), pp < /j.ydbio >. <hal > HAL Id: hal Submitted on 3 Dec 2014 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 Pax8andPax2arespecificallyrequiredatdifferentstepsofXenopuspronephros development Abstract Xenopus hnf1blhx1osr2, dvl1 sfrp3 XenopusPax2/8hnf1b 1

3 INTRODUCTION Xenopus Xenopus Xenopus Xenopus lhx1pax8osr12 exvivo wt1 2

4 Pax2/5/8 pax2 Pax8 pax2/pax8/ pax2pax8 Xenopuspax2a no isthmus pax2a/pax8 Xenopuspax8 pax2 lhx1 pax2pax8xenopus Xenopus hnf1b, osr2lhx1 dvl1sfrp3 3

5 MATERIALSANDMETHODS Xenopusembryosandmicroinjections XenopuslævisXenopus Xenopuslævis pax8 pax8 pax8 Explantsdissectionsandincubation 4

6 RealtimequantitativePCR P<0.005P<0.001 Insituhybridization insitu insitu Immunodetection 5

7 BrdUincorporation insitupax8 pax8 tp<0.005p<0.001 RESULTS Pax8andPax2tadpolemorphantsdisplaydistincttubulephenotypes pax8pax2xenopusin vivo pax8pax2 pax2pax8 6

8 in situpax2 smp30 outerdensefibre3(odf3) evi1 pax2 odf3 smp30 evi1 pax2 odf3 smp30 evi1 slc4a4 NBC1 slc12a1 NKCC2atp1b1 clcnkb1claudin19 atp1b1 clcnkb1 nephrin wt1 wt1nephrin pax8 pax2 nephrinwt1in situ wt1 nephrin 7

9 wt1nephrin pax2 pax8 pax8pax2 Pax8 but not Pax2 is required for tubule marker genes expression and anlage formationattheearlytailbudstage lhx1hnf1bpax2hrt1evi1lhx1hnf1b pax2 Hrt1 evi1 insitu lhx1 hnf1bpax2hrt1evi1 hrt1 hx1evi1 8

10 invitropax8 pax8 pax8 pax8gr lhx1 Hnf1bexpressioninthekidneyfield(KF)isunderthecontrolofPax8 insitu lhx1osr2hnf1blhx1osr2 lhx1;osr2 hnf1b hnf1b osr2 lhx1 hnf1b hnf1b invitro hnf1b Hnf1b hnf1b 9

11 Pax8GR hnf1b hnf1b hnf1b hnf1b Pax8knockdownleadstoanincreaseofbloodandendothelialgenesexpression domainsinthedorsolateralplate(dlp) myodmlcfoxf1 scl lmo2, msr,flk1 myodmlc pax8 insitupax8in situ pax8foxf1mlc pax8 mlc foxf1 pax8 pax8 msrflk1lmo2scl msrflk1 lmo2scl insitupax8lmo2lmo2 Lmo2pax8 pax8 10

12 lmo2 lmo2 insitu lmo2 pax8 Pax8knockdownleadstocellproliferationdefectthatisrescuedbyactivationof thewnt/cateninpathway pax8 insitu Xenopus Pax8lossoffunctionaffectsexpressionofseveralcomponentsofthewntpathway wnt4, wnt5a, wnt8, wnt11b, wnt6, wnt9a, 11

13 wnt9b, crescent, dkk1, dkk3, sfrp1, sfrp2, sfrp3, sfrp5, sizzled dvl1,dvl2dvl3 lhx1pax8 sox17betamyf5,. dvl1 sfrp3 sfrp3 dvl1 in situ dvl1sfrp3dvl1 dvl1 insitu sfrp3 insitu dvl1sfrp3exvivo dvl1 dvl1 sfrp3 sfrp3 sfrp3 dvl1sfrp3 DISCUSSION Xenopus hnf1b 12

14 Xenopus smp30, slc4a4, atp1b1 odf3claudin19 Clcnkb1 atp1b1 pax2 Pax8insitu pax2 pax2 pax8pax8 rubyxenopustropicalis pax8. pax8pax2 pax8/ pax2/pax8/pax2 pax8 hnf1ba/b hnf1ba/b hnf1bxenopus hnf1b Pax2Pax8 Xenopus pax2/wt1 wt1 nephrinwt1 pax2a 13

15 pax8pax2wt1xenopus Xenopus dvl1 sfrp3 wnt4 sfrp2 wnt5a Xenopusdvl1 sfrp3 sfrp3 sfrp3 Grg4Grg5 14

16 pax8 sfrp3 sfrp3 dvl1 hnf1b hnf1b dvl1sfrp3 pax8 pax8 pax8 Xenopus fli1,msr,gata2, scllhx1gata3 lmo2,gata2,flk1,msr pax2a 15

17 scl pax2pax8 Xenopus hnf1b Acknowledgments References 16

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24 FIGURELEGENDS Figure1:TadpolesdepletedforPax2orPax8displaypronephricdefects (A) (B) (C) Figure 2: Pax 8 and Pax2 loss of function differentially affects tubule but not glomusmarkergenesexpressionatlatetailbudstage (A,B) in situ insitu clcnkb1 (C)insitu wt1nephrin (D)wt1nephrin. wt1nephrin 23

25 Figure 3: Pax8 but not Pax2 loss of function inhibits tubule marker genes expressionandpronephrictubuleanlageformationattheearlytailbudstage (A)insitu insitu lhx1 hnf1b pax2 hrt1 evi1 (B) Figure 4: Inhibition of lhx1 expression in response to MoPax8 is rescued by expressingpax8 lhx1insitu lhx1 Figure 5: Hnf1b but not osr2 and lhx1 expression is inhibited in the KF of Pax8 morphantatneurulastage A Insitu lhx1 osr2hnf1b insitu Hnf1b B lhx1 osr2 hnf1b lhx1 osr2hnf1b 24

26 P Figure 6: Pax8 ectopic expression is sufficient to upregulate hnf1b in ectoderm animalcapsinabsenceofproteinsynthesis ABhnf1bA pax8 Hnf1b Bpax8GR hnf1b hnf1b P Figure7:Expressionofsomitic,lateralplate,bloodandendothelialmarkergenes inpax8morphants (A) myodmlc foxf1 insitu myod mlc foxf1(b)scllmo2msr flk1 insitu pax8lmo2 scl lmo2,msrflk1 insitupax8lmo2 lmo2 pax8 pax8 (C)lmo2 insitulmo2 25

27 Figure8:MoPax8knockdownresultsincellproliferationdefectintheKFthatis rescuedbyactivationofthecanonicalwntpathway A insitu insitu pax8 pax8 B C PP Figure 9: Pax8 regulates the expression of components of the wnt signaling pathway A ) dvl1 sfrp3 crescent,dkk1,dkk3,sfrp1,sfrp2,sfrp5 sizzledwnt4, wnt5a, wnt8, wnt11b wnt6, wnt9a, wnt9b dvl2 dvl3 BInsitudvl1 insitu Dvl1 C, D) dvl1 C sfrp3 D (pax8,pax8vp16,pax8do) dvl1 P 26

28 Supplementarydata Figure S1: MoPax8 and MoPax2 block endogenous embryonic Pax8 and Pax2 expression,respectively (A) (B) pax2pax8 FigureS2:PronephricphenotypeinducedbyinjectionofMoPax2.2 insitu (A) (B) insitu clcnkb1 wt1nephrin Figure S3: Pronephric phenotype induced by coinjection of MoPax8 and MoPax8.B insitu 27

29 (A) (B) in situ pax2, odf3, smp30, lhx1, hnf1b pax2 hrt1 evi1 wt1nephrin FigureS4:Hnf1bbutnotosr2andlhx1expressionisinhibitedintheneurulaKFin responsetothecoinjectionofmopax8andmopax8.b In situ lhx1 osr2 hnf1b insitu Hnf1b FigureS5:Characterizationofthekidneyfieldexplants. lhx1 pax8 myf5 sox17beta cytokeratin Pax8 lhx1sox17beta myf5 cytokeratin PPP - Pax8 is a major regulator of the first steps of pronephric development - Pax2 is required for the expression of clckn, a tubule differentiation marker gene - Pax8 is required for the expression of hnf1b in the kidney field - Pax8 controls expression of the wnt pathway genes dvl1 and sfrp3 in the kidney field - Pax8 is required for cell proliferation of pronephric precursors 28

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