PROGRAMME DE STAGE D’INITIATION A LA RECHERCHE AU 1ER CYCLE
SIRI
Université de Lyon/Université d’Ottawa
Appel à candidature 2017
Title
The extracellular matrix collagen XV as a new player of
motor axon development: a functional study using zebrafish
Supervisor-­‐s
Surname RUGGIERO
First Name FLORENCE
Director of IGFL
Email florence.ruggiero@ens-­‐lyon.fr
Phone 00 33 4 26 73 13 58
Surname BRETAUD
First Name SANDRINE
Lecturer at Université de Lyon 1
Email sandrine.bretaud@ens-­‐lyon.fr
Phone 00 33 4 26 73 13 55
Institute
Institut de Génomique Fonctionnelle de Lyon (IGFL)
ENS de Lyon, CNRS UMR 5242, Université de Lyon
Lab
Matrix Biology and Pathology Team
Context
From spinal cord exit, motor axons are guided by attractive and
repulsive cues along a stereotyped path and navigate with remarkable precision to their muscle target. Zebrafish has been
particularly instrumental in identifying specialized cells and
extracellular environments critical for motor axon pathfinding
(Beattie, 2000). The myotome provides positional information
to guide primary and secondary motoneuron axons along appropriate paths (Panzer, 2005). Slow muscle precursors, named
adaxial cells, which are located adjacent to the notochord before somitogenesis, represent an important source of various
extracellular matrix (ECM) components of the motor path. As
such, the glycoprotein tenascin-C (Schweitzer et al, 2005) and
chondroitin sulfate proteoglycans (CSPGs; Bernhardt and
Schachner, 2000), both expressed by adaxial cells, have been
recognized as important short-range guidance cues for axon
outgrowth. Nevertheless, little is known about the identity of
these extracellular matrix cues and the underlying mechanisms.
Abstract/Objectives
Collagen XV is a component of the basement membrane, a
specialized structure present at the interface between tissues or
surrounding individual cells, as the motor neurons in peripheral
nervous system. This collagen, and its structural homologue
collagen XVIII, were found to control axonal guidance in the
two main invertebrate organism models, the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans.
However, the mechanisms by which collagen XV takes part to
this process remain elusive. To address this important question,
we propose to use zebrafish, our favorite animal model.
We have identified in the zebrafish genome two paralogs of the
human COL15A1 gene, col15a1a and col15a1b, that display
distinct expression patterns (Pagnon-Minot et al, 2008; Bretaud
et al, 2011). COLXV-A was expressed in the notochord and
played a role in muscle differentiation by interplaying with Shh
signaling (Pagnon-Minot et al, 2008). We recently demonstrated that col15a1b expression and subsequent collagen XV-B
deposition and organization in the motor path ECM depend on
a previously undescribed two-step mechanism involving
Hedgehog/Gli and unplugged/MuSK signaling pathways. Lossof-function using a mutant line or gain-of-function by overexpressing col15a1b in slow muscle fibers both provoke pathfinding errors of motoneuron axons (Guillon et al, 2016).
The present project aims at specifically understanding the
mechanism of action of COLXV-B on neurite formation at the
cell level. To address this question, we combine different
approaches (biochemistry, cell culture, imaging, cell sorting,
development) and utilize motor neurons isolated from
developing embryos. The motoneurons will be isolated
from a zebrafish fluorescent transgenic line by FACS and
their behaviour as well as their capacity to extend neurites
will be investigated using video time-­‐lapse microscopy in
presence or absence of zebrafish COLXV-­‐B produced re-­‐
combinantly. To determine whether COLXV-­‐B alone can act
as a repulsive or attractive contact cue, the neurite behavior
of isolated motoneurons will be also analyzed on COLXV-­‐B
micro-­‐patterned tracks by printing the protein on the
tracks or only on the track borders (in collaboration with M.
Balland, University of Grenoble). This work will contribute to
a better understanding of the role of the extracellular matrix in
the development of neuromuscular system, and more specifically to decipher the mechanisms by which collagen XV regulates
directly or indirectly motoneuron axon pathfinding.
Bibliography
Beattie CE (2000). Control of motor axon guidance in the zebrafish embryo.Brain Res Bull. 53,489-500.
Bernhardt RR, Schachner M (2000). Chondroitin sulfates affect the formation of the segmental motor nerves in zebrafish embryos. Dev Biol. 221,
206–219.
Bretaud S, Pagnon-Minot A, Guillon E, Ruggiero F, Le Guellec D
(2011). Characterization of spatial and temporal expression pattern of
col15a1b during zebrafish development. Gene Expr Patterns, 11:129-34.
Guillon E, Bretaud S, Ruggiero F (2016). Slow muscle precursors lay
down a collagen XV matrix fingerprint to guide motor axon navigation.
J. Neurosci. 36, 2663-2676.
Pagnon-Minot A., Malbouyres M., Haftek-Terreau Z., Kim R, Sasaki
T., Thisse C., Thisse B., Ingham P.W., Ruggiero F. and Le Guellec D.
(2008). Collagen XV, a novel factor in zebrafish notochord differentiation and muscle development. Dev. Biol. 316:21-35
Panzer, J. A., Gibbs, S. M., Dosch, R., Wagner, D., Mullins, M. C., Granato,
M. and Balice-Gordon, R. J. (2005). Neuromuscular synaptogenesis in wildtype and mutant zebrafish. Dev. Biol. 285, 340–357.
Schweitzer J, Becker T, Lefebvre J, Granato M, Schachner M, Becker CG
(2005). Tenascin-C is involved in motor axon outgrowth in the trunk of
developing zebrafish. Dev Dyn. 234, 550–566.
Location
Institut de Génomique Fonctionnelle de Lyon
ENS de Lyon - CNRS UMR 5242 - Université de Lyon
46, allée d'Italie
69364 Lyon cedex 07
Duration
3 months
Both (lab meeting are exclusively in English and IGFL is an
Language
(French/English/Both) international institute hosting dozen different nationalities)