Max-Planck-Institute for Molecular Genetics Development and Disease Group FG Mundlos Development "%% L & Disease Tyrosinkinase Ror2: Funktion, klinische Variabilität und Interaktionsnetzwerk Brno, 17.1.2008 patterning, organogenesis, differentiation •• • • • v #•*• • • • • • •*• • • •*•• • undifferentiated • •# • # cells • # • • • dHAND r GU3 -| patterning: gene expression changes I wt wt patterning, organogenesis, differentiation dHANDrGH3-i A B : patterning: gene expression changes organogenesis: morphological shaping patt erning, or ganogenesis, differentiation dHAND A B .^HÉ^^^^fl^Bb^» •• * I -*4 %5> ĚĚtm ••••:•> ••••:•••; I wt Wt '** • • • •• W^ •• • •••:*• tf^* ;> ^^H undifferentiated ••••:•••••• —*\- M cells • • • • • X • •••Wt* j patterning: •■■ •••IL" • gene expression changes Ä#~ J**^^ ^ »V • •• # \. • •!•*• aV organogenesis: ••-■■ ••V« •4:s:Vr* morphological -• "S* t ■ • shaping • •• # differentiation: emergence of new cell types within the organ patterning, organogenesis, differentiation 1 2 -1—i—■—r 3_____4 t—'—r 5___ 6 7 8 9 tO 11 12 13 t4 15 16 17 18 19; t—'—i—'—i—'—i—'—i—■—i—■—i—■—i—■—i—'—i—■—r"- Mi ir\\ /TV .f jA 8 A Pr* *♦' D "►Di A P ♦ D V ♦ D AER (Fgfô) Ill Hand malformations Mutations in Ror2 cause dominant Brachydactyly type B (BDB) ...and recessive Robinow syndrome (RRS) Ror2 BDB IG-like Kringle Cystein-rich Transmembrane Tyrosinkinase Proline-Serine-Threonine-rich C182X R189W R184C R205X R366W R396X Q502X 1740-1774del35 N620K W720X RRS Ror2 Deficient Mice (delEx2) as a Model for Recessive Robinow Syndrome wt Ror2-/- wt Ror2-/- (Schwabe et al. Dev Dyn 2004) Chicken Ror2 Constructs for Retroviral Overexpression «a 4v ^ % ^ ROR2 cRor2 R433X cRor2 E459X-c/hRor2 1374insA- cRor2 Q494X- X X •a m CM CO CM CO T— co Q m < i/) c ČO O) CO T— m Q m i i i ▼ *>„ "*/ Tít X CM O m a 03 QĹ w °% % °% % % % B 100kDa— 70kDa— 50kDa— ß-actin Retroviral Overexpression of Genes in the Chick Embryo SD LTR SA SA gag pol env subgroup variable Kpn] rtŕion Sa|| Clal í LTR ■ """'■■■ Clal í (i) ATG SloP , Slop gflg I pot I AAAA ' ' ' J- J I RT ÍN MA plO CA NC PR (Hi) ATG oN Slop env V X SU TM v N -^ <*" s N 's AAAA ATG ATG s'"pi f sene X Stop AAAA ^ prothtn x from Logan and Tabin, 1998 Injection into Limb Fields Stage 10 Limitations of the RCAS system: -maximum insert size ~2.4 kb -RCAS infects any replicating cell —> no cell-type specific overexpression -variable efficiency of virus delivery, targeting of injection from Logan and Tabin, 1998 Overexpression of Ror2 Mutants in Chick Limb Field B R433X c o o o o E459X r^ k*. 1374insA /XH Q494X Genotype / Phenotype correlations: BDB BDB only frameshift-mutations ■► frameshift and nonsense-mutations RRS Mutation C1324T -> R442X unaffected Robinow syndrome + Brachydactyly Mischphänotypen sind abhängig von Position und Art der Mutation TM ■1320 -1330 ■1340 ■1350 ■1360 ■1370 ■1380 1390 -I-1400+ TK Dominant BDB 1321-1325del5 -> FS Recessive Robinow syndrome +Brachydaktyly (not dominant BDBU) C1324T -» Stop 1367insC -> FS splice site —> FS 1398insA -> FS 1399insT -> FS BDB BDB + Robinow features! Intracellular localization of Ror2 wt and mutant protein wt 1504OT(RRS) 1860T>A(RRS) 2160G>A(RRS) 22460A (BDB) del1321-1325 (BDB) 1396insA (BDB) 13240TÍRRS/BDB) ROR2 BAP31 TM ■1320 ■1330 ■1340 1350 ■1360 1370 1380 1390 rRTÜÖ" Difference BDB /RRS BDB RRS 1321-1325del5 -> FS C1324T -> Stop 1367insC -> FS splice site —» FS 1398insA->FS 1399insT -> FS RRS C1504T -> Stop membrane localization intracellular retention dominant-negative effect by truncated forms of Ror2 loss-of-function probably unfolded protein response Die Ror2-Bry (W749X) Maus Aris Economides / Regeneron Inc., Tarrytown, NY WT Ror2 hry/hry Ror2 Ror2-Bry W749X v ^ v^»> No early patterning defect in the Ror2-Bry mouse Shh wt bry/bry Fgre wt bry/bry 10.5 , 12.5 13.5 r ) wt bry/bry HoxDll Phenotype part 1: Cartilage condensation in the Ror2-Bry mouse wt bry o Q. ■o lO ei o Q. ■a lO co o Q. ■o lO HE/AlcianBlue. Cartilage condensation: cell recruitment Pulse-Chase experiment: lh BrdU labeling (Pulse), 10h unlabeled uridine (Chase) bry 14,5 vswt 13,5 tip = surrounding sox9 distal condensation = p2/3 positive cells cells ' n r D heterozygous ■ brachy — n <í c n á 0,46 o u " n 0,37 íu n ^ 0,32 gí u,° o a 02 0,27 n i 0,19 0 - sum Pathways in phalangeal condensation dorsal ventral whole mount in situ hybridization bry wt skeletal preparations hindlimb forelimb IS) • • • • •• • •j IVJ Ol w Ol ■&■ Ol N % - % A* % 3 ■a o w Ol Q. ■a o ö 5c* a-a gtS" 3* QfQ O I öd o C/5 Deregulation of Sox9 in distal condensations in situ hybridizations - FL 13,5dpc whole mount %*' section antibody staining Sox9 (Alexa546) / DAPI real time per - FL 13,5dpc % * • • Bmp Signaling: Ror2-BDB and Gdf5 show genetic interaction crossing ror2-brachy with gdf5-brachypodism mice middle fingers of newborn mice bry het / gdf5 wt bry het / gdf5 het bry horn / gdf5 het f* r+\ ♦. ..♦ / •♦. ♦.......•• \..T./ -. * • - bry wt / gdf5 het bry het / gdf5 het bry het / gdf5 horn bry horn / gdf5 hom Phenotype part 2: decreased proliferation in flanking mesenchyme proliferation assay, BrdU incorporation, 13,5 dpc 0,35- 0,3 IS 0,25 i_ C O 0,2 I 0,15 O 0,1 Q. 0,05 25% 10% wt bry decreased proliferation due to decreased expression of CyclinDl wt bry HE/AlcianBlue Pathways in phalangeal condensation dorsal ventral Reduced Fgf signalling in the Ror2-Bry mouse Fgf8 r s-\ r ( \ J ^ Ä AER ■■■■-■■■'■ ^M r Sproutyl > ) Sprouty4 ES^S^S^B - Pathways in phalangeal condensation dorsal canonical Wnt proximal distal AER: Fgfs ventral Upregulation of canonical Wnt signalling ?? TOPGAL bry/bry ^ Ror2-Bry \ ® s MA/nľ) / Tbiup) 1 J / r\ The brachydactylies: a syndrome family approach MPI Berlin: Stefan Mundlos Florian Witte Wibke Schwarzer Norbert Brieske Britta Hoffmann Kathrin Seidel Regeneron Ine, NY: Aris N. Economides Regina Raz Thank You Vitezlav Briya Karlsruhe University: Alexandra Schambony