Identifying new functions of the EGF/ EGFR pathway through tissue-speciﬁc recombination Silvan Spiri, Louisa Mereu, Matthias Morf, Alex Hajnal Institute of Molecular Life Sciences, University of Zurich LIN-3 EGF LET-23 EGFR AC P4.p P5.p P6.p P7.p P8.p P3.p Basement membrane 1° LIN-3 EGF LET-23 EGFR AC P4.p P5.p P6.p P7.p P8.p P3.p Basement membrane A) EGF/ LIN-3 dependent induction of the vulval precursor cells. VulE VulE VulF VulF AC VulF VulF VulE VulE C) The AC directly promotes dorsal lumen formation during vulva morphogenesis. Ventral nerve cord Basement membrane 2° 2° 1° 1° 1° 1° UNC-6 FOS-1 ? ? B) The anchor cell (AC), guided by Netrin/ UNC-6 and unkown VPC guidance cue`s, breaches the basement membrane after the VPCs divided the second time. FRT GFP let-23::gfp let-23 let-23::gfp FRT Protein kinase domain FRT GFP E) Two FRT sites, ﬂanking the sole kinase domain of egfr/ let-23, along with a GFP sequence are inserted into the endogenous egfr/ let-23 locus. Upon Flp expression the kinase domain is excised resulting in in- active receptor and loss of GFP signal. Tool to induce a temporal or spatial speciﬁc knock-out of egfr/ let-23: ! " VulE VulE VulF VulF AC VulF VulF VulE VulE utse utse utse utse utse LET-23 EGFR LIN-3 EGF uv1 cell fate uv1 utse uv1 D) EGF/ LIN-3 is expressed in the 1° vulval cell lineage after vulva induction and is necessary to specify the uterin uv1 cell fate. Introduction Model Summary VulE VulE VulF VulF VulF VulF VulE VulE AC LET-23 EGFR LIN-3 EGF EGFR is expressed in the AC from the onset of vulval mor- phogenesis, securing dorsal lumen formation by stabilizing cytoskeleton components during lumen opening 1) EGFR is expressed in the AC during vulval morphogenesis 2) AC mispositioned in AC EGFR-KO animals during L4.1-2 3) F-actin in AC is disorganized during L4.2-3 in AC EGFR-KOs 4) In a sensitized background BM breaching during morpho- genesis is aﬀected in AC EGFR-KO animals 5) Narrow dorsal lumen upon AC EGFR-KO during L4.4 6) KO of lin-3 in the VPCs or let-23 in the AC leads to egg laying defective adults let-23 egf X egfrX 3) KO of egfr/ let-23 in the AC leads to more variability in AC alignment during L4.1 and L4.2 4) KO of egfr/ let-23 in the AC leads to disorganized F-actin network in L4.2 6) KO of egfr/ let-23 in the AC leads to a decreased dorsal lumen diameter during L4.4 7) KO of lin-3 in the VPCs or let-23 in the AC leads to egg-laying defectie animals Ventral nerve cord Basement membrane 2° 2° 1° 1° 1° 1° UNC-6 FOS-1 ? ? Vulval development and morphogenesis: let-23::gfp Ac::Flp; Nomarski Ctrl; Nomarski let-23::gfp Anchor cell Alignment L4.1 ns p=0.0086 p=0.0005 0 1µm 2µm 3µm 4µm Ctrl Ac::Flp Ctrl Ac::Flp KO Anchor cell Alignment L4.2 p=0.0024 ns p=0.0120 Ctrl Ac::Flp Ctrl Ac::Flp KO 0 1µm 2µm 3µm 4µm LET-23::GFP signal is lost upon AC speciﬁc KO of egfr/ let-23 resulting in a more variable position of the AC during L4.1 and L4.2: (A) schematic of AC speciﬁc KO of egfr/ let-23. (B) Example images of AC speciﬁc KO animals during L4.1 on the left and control animals on the right. Upper row Nomarski images, bottom row LET-23::GFP. Scale bar represents 5 µm, arrowhead indicates the AC. (C) Quantiﬁaction of AC alignment relative to the vulval midline during L4.1 and L4.2. A) B) C) Ac::Flp; Nomarski Ctrl; Nomarski AJM-1::GFP AJM-1::GFP AC speciﬁc KO of egfr/ let-23 leads to a smaller dorsal lumen diameter during L4.4: (A) Example images of AC speciﬁc KO animals during L4.4 on the left and control animals on the right. Upper row Nomarski images, bottom row AJM-1::GFP to mark the apical junctions. (B) the dorsal lumen diameter was determed by measuring the inner diameter of the vulF toroid using the vulF/ vulE apical junctions. A) B) L4.4 Dorsal lumen diameter p=0.0002 Ctrl n=22 Ac::Flp n=22 9µm 8µm 7µm 6µm 5µm 4µm 3µm 2µm 1µm 5) KO of egfr/ let-23 in the AC leads to decreased base- ment membrane breaching during morphogenesis in a sensitized background A) B) egfr/ let-23 KO increases failure