1. 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

2. 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

3. 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

4. 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

5. 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

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

8. Shh Signal pathway
Mukhopadhyay, S. et al. Cell 2013; 152:

9. 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

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

11. 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

12. 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 Vil. 7: )

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

14. Transfection reagent Dharmafect suppresses pathway activation

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

16. Reduced Rgnef expression suppresses Shh pathway activation

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

18. 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)

19. 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

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