How to grow an extra eye?
A story of the protein Asporin, IGF signaling and the African clawed frog.
Kristin Luehders, Noriaki Sasai, Hitoshi Hiura, Tara Brah and Shin-ichi Ohnuma
UCL Institute of Ophthalmology, UCL, 11-43 Bath Street, London EC1V 9EL, UK
INTRODUCTION
RESULTS
During early eye development, a defined region in the anterior neural
plate develops into the eye field, which is marked by the expression of a
series of eye field transcription factors (EFTFs) such as Otx2, Pax6,
Rx1 and ET. These transcription factors then transcribe a variety of
downstream eye-specific genes, which eventually results in a fully
developed eye. It has been repeatedly shown that overexpression of the
EFTFs tends to induce “ectopic” eyes during early development. Little is
known, however, about the mechanism of initial determination of
the eye field location through secreted proteins.
Otx2
Noggin
ET, Rx1,
1) ASPN Overexpression Phenotype
Eye
Pax6 etc.
Neural Induction
Fore/Midbrain specification
Eye field specification
(adapted from Zuber et. al 2003)
Asporin (ASPN) is a member of the small leucine-rich proteoglycan
(SLRP) family of secreted proteins. SLRPs are biologically active
components of the extracellular matrix, but have recently been also found
to interact with various signalling pathways which affect crucial cellular
processes such as growth, differentiation and survival. Asporin was first
identified in human articular cartilage and has since been associated with
osteoarthritis and cancer.
•  Enlarged (B, B’) and/or extra ”eye”-like
tissue (C, C’) found in ASPN injected
tadpoles
•  Histological analysis revealed retina-like
layered morphology of these tissues (E, F)
2) ASPN changes expression of eye specific transcription factors
a)
b)
We found that Asporin is expressed in the
developing African clawed frog Xenopus laevis
and overexpression of ASPN leads to tadpoles
with - what looks like - “extra eyes”.
Asporin has the ability to change
expression patterns and levels of several
developmental factors as seen in
wholemount in situ hybridisation (a)
and RT-PCR analyses (b):
OBJECTIVE
•  Sections of ectopic “eyes” stained
positive for eye specific markers:
- Lens (β-crystallin)
- Müller Glia (Glutamine Synthetase)
- Amacrine and Horizontal cells
(HuC/HuD)
Xenopus laevis frog
•  It expands the expression of eye
field transcription factors Rx, Pax6
and Otx2 (B, D, F)
² Perform a detailed characterisation of the eye phenotype found in
Xenopus embryos following Asporin overexpression
•  It induces several neural forebrain
markers (NCAM, Sox2)
² Elucidate the molecular mechanism, through which Asporin
induces these phenotypes
•  It down regulates mid/hind brain
markers like En2, Krox20 (J, L) and
Slug
METHODS
•  Microinjection of mRNA (e.g. Asporin, IGF2, DN-IGF-1R) into one or
more animal blastomeres of 4-8 cell embryo
•  Animal Cap Assay: Following mRNA microinjection, embryos were
grown to stage 9.5-10 and the so called “animal cap” dissected and
processed for PCR analysis
3) ASPN elicits this functions by activating IGF-1R?
c)
d)
e)
•  Paraffin embedding and sectioning of tadpoles, followed by H&E
staining
•  Embedding of tadpoles in OCT compound and cryosectioning,
followed by Immunohistochemistry
•  Whole-mount in situ hybridisation with DIG labeled riboprobes
•  Co-immunoprecipitation of Asporin and IGF-1R receptor using
transfected HEK293 cells
In vivo methods Overview
mRNA
injection
Analyze
Phenotypes
at St. 45
Grow embryos
4-cell embryo
•  Cryo- or paraffin
sectioning
•  Immunohistochemistry
•  In situ
Hybridisation
PCR
Analysis of
Animal cap
RNA
Dissect
animal cap
•  S t u d i e s u s i n g a
dominant-negative
form of the IGF-1R
receptor supports
Asporin acting via
IGF-1R
•  P r e l i m i n a r y r e s u l t s
indicate that Asporin
also affects other
signaling pathways:
Nodal (e), Wnt and
BMP (not shown)
CONCLUSIONS
u  Asporin is a novel molecule involved in eye induction and development
Animal Cap
assay
St.9.5- 10
embryo
•  C o - i m m u n o p r e c i p i t a t i o n
experiment,
showed that
Asporin binds to the
IGF-1R receptor
u  It seems to play an important regulatory role upstream of eye field transcription
factors
u  Asporin is likely to act through the IGF-1R receptor to which it binds
u  Other signaling pathways are likely to be also affected by Asporin during eye
development