24th MAMMALIAN GENETICS AND DEVELOPMENT WORKSHOP KAYVAN HAKIM-RAD, ANJU PAUDYAL, JENNIFER MURDOCH Investigating the molecular function of Tulp3 interacting protein Rgnef in Shh signalling

Overview Introduction to Tulp3, Rgnef and Shh signalling Results Early embryonic expression of Rgnef Shh LII cell assay relating to Rgnef expression Preliminary data – Rgnef cell localisation Summary

Hitchhiker: Tulp3 mutant with NTDs Exencephaly Limb defects The ENU mutagenesis-derived hitchhiker mouse mutant exhibits preaxial polydactyly, exencephaly and spina bifida as well as abnormal dorso-ventral patterning of the spinal cord. This phenotype is derived from a single base pair transversion in the Tubby-like-protein 3 gene (Tulp3) resulting in a splicing defect that almost completely inactivates protein production So this mutant came from an ENU recessive 3-generation screen. The reason we first became interested in it is because of these phenotypes; sp bif and ex, and limb defects. Also has polydactyly. It demonstrates the NTDs exencephaly and spina bifida, and also has this appearance of caudal oedema which is actually due to expansion of the neural tube. It also has limb defects including preaxial polydactyly, and abnormal morphology of the first digit, which is why we gave it the name hitchhiker. Neural tube expansion Spina bifida

Neural tube dorso-ventral patterning Two balancing signals Dorsalizing signal- BMP from roof plate and surface ectoderm Roof plate Dorsal interneurons Dorsal Surface ectoderm Neural tube V0 ventral Motor-neurons Ventral V3 ventral Notochord Ventralizing signal- SHH from floor plate and notochord Floor plate

Hitchhiker: Tulp3 mutant with NTDs Dorsalizing signal- BMP from roof plate and surface ectoderm Expansion of ventral markers Tulp3 negative regulator of the Shh signal pathway The result with Shh prompted us to look at other markers of dorso-ventral patterning in the neural tube, since Shh is itself a key regulator of patterning in the neural tube. These are images of immunofluorescence staining on transverse sections of the caudal region of the neural tube, in 10.5 day embryos. We looked at a whole range of markers of dorso-ventral patterning in the neural tube, these are just a few examples; all the ventral markers we looked at showed expansion into more dorsal regions of the neural tube, in the mutant embryos. Correspondingly, the more dorsal markers were reduced, such that their ventral boundaries of expression were pushed further dorsally. So the hitchhiker mutants exhibit a general ventralization of the caudal spinal cord. Ventralizing signal- SHH from floor plate and notochord

Shh Signal pathway Huangfu, D. et al. Development 2006;133:3-14

Shh Signal pathway Mukhopadhyay, S. et al. Cell 2013; 152: 210-223

Rgnef interacts with Tulp3 LEXA Tulp3 GAL4 Tulp3 Yeast 2-hybrid screen (x2) Rgnef - guanine nucleotide exchange factor Axonal branching Synapse formation Dentritic morphogenesis Focal adhesion formation Cell motility Regulate NEFL expression Binds depleted GDP bound form of RhoA to then facilitate its transfer to a GTP bound form (van Horck et al., 2001). Yeast 2-hybrid screen: full length Tulp3, mouse embryonic brain library Trim71 demonstrated to function as an E3 Ubiquitin ligase Transcript expression regulated by miRNA Trim71 binds to proteins involved in miRNA processing Involved in timing of cell fate in C. Elegans Induced by Shh expression (bead implants, chick) Direct role - Interacts with RhoA to induces cytoskeletal contractions and inhibits neurite growth Binds to microtubules - role in intracellular trafficking Binds scaffolding protein JIP-1 leading to JNK activation (without GDP/GTP exchange) (Meyer et al., 1999). Known and predicted protein interaction diagram generated from STRING database Jensen et al. Nucleic Acids Res. 2009, 37(Database issue):D412-6 9

Rgnef is expressed in neural tube of early mouse embryos E10.5 mouse embryo whole mount in-situ hybridisation

Rgnef neural tube and floor plate expression is consistant with a role in neural tube development Rgnef Expression Shh Expression Neural Tube Notochord Floor Plate

Shh light II cells cell based shh pathway activity assay Day1 siRNA transfection Day2 Serum Free media Day3 Purmorphamie activation Transfection reagent Purmorphamine siRNA Smoothened Rgnef transcripts ? Shh pathway Day4 Dual-Glo Luciferase assay (Promega) Gli protein Ciliogenesis 8XGli BS promoter  luciferase reporter Cell number  Renilla luciferase Gli-responsive Firefly luciferase reporter (H. Saski et al., Development 1997 Vil. 7: 1313-22)

Transfection reagent Dharmafect suppresses assay window *** * N=12

Transfection reagent Dharmafect suppresses pathway activation

Dharmafect does not affect ciliogenesis Fluorescent stained images cell nucleus (DAPI) cilia (N-acetylated tublin) untreated Dharmafect treated

Reduced Rgnef expression suppresses Shh pathway activation

Increased Rgnef expression activates Shh pathway ** N=12 N=12

Rgnef HA expression in NIH 3T3 cells Preliminary results Rgnef HA expression in NIH 3T3 cells untransfected pRgnefHA Fluorescent images cell nucleus (DAPI) cilia (α N-acetylated tublin) pRgnef HA (α HA tag)

Summary Indication of Rgnef neural tube and floor plate expression is consistant with a role in DV Rgnef expression changes relate to Shh pathway activation Future plans Rgnef cell localisation in relation to cilia Mass spectrometry analysis of Rgnef protein pull-down, before and after pathway activation Binding protein and phosphorylation sites

Jenny Murdoch, Christopher Wilkinson, Vicki Patterson, Anju paudyal Thanks to Jenny Murdoch, Christopher Wilkinson, Vicki Patterson, Anju paudyal James Briscoe & W H Moolenaar