Hormones, Simulated Microgravity and Hypergravity affect Bone and other Physiological Systems ...

259 Pages·2015·11.46 MB·English

University of Liège

Faculty of Science

Groupe Interdisciplinaire de Génoprotéomique Appliquée
Département des Sciences de la Vie
Laboratoire d’Organogénèse et Régénération

Hormones, Simulated Microgravity and
Hypergravity affect Bone and other
Physiological Systems in Zebrafish

Larvae

By

Jessica Aceto

A thesis submitted in fulfillment of the requirements

for the degree of Doctor of Philosophy (Life sciences)

Academic year 2014-2015

Thesis Supervisor:
Professor Marc Muller
Acknowledgement - Remerciements
Je voudrais remercier le Professeur Martial de m’avoir accueillie au sein de son laboratoire,
pour ses encouragements et sa positivité.
Je remercie tout particulièrement mon Promoteur Marc Muller de m’avoir donné l’opportunité
de réaliser ce doctorat au sein de son équipe et d’avoir cru en moi du début jusqu’à la fin de la
concrétisation de cette thèse. De m’avoir permis de m’ouvrir au monde par les congrès et
collaborations qui m’ont énormément appris scientifiquement, permis de m’affirmer et de
prendre confiance en moi.
Je tiens aussi à remercier les membres de l’équipe zebrafish, tous ces collègues qui m’ont
permis de réaliser la transition entre le monde des études et le monde professionnel. Un peu
trop nombreux pour tous les citer. Mais chacun à leur manière m’ont apporté, au quotidien, un
nouveau point de vue sur la manière de travailler et d’évoluer au sein du labo. Une attention
toute particulière pour remercier Marie Winandy qui a toujours pu trouver des solutions pour
mes commandes poissons assez particulières ou de dernière minute. De même, un grand merci
aux étudiants que j’ai eu l’occasion d’encadrer et avec qui j’ai pu grandir en expérience aussi.
I would like to thanks all the collaborators from the ESA MAP projects. Especially, Dr. Peter
Aleström, Dr. Rasoul Nourizadeh-Lillabadi from Oslo and Dr. Jack van Loon from
Amsterdam, for making me so welcome in their lab, their good advices and the rich scientific
experience. As well Dr. Janette A.M. Maier and Dr. Massimo Mariotti from Milan, to
welcome and integrate me in their lab.
De même, j’adresse un merci tout particulier aux bioinformaticiens Raphaël Marée, Olivier
Stern et Nathalie Jeanray, ainsi qu’à Nadia Dardenne, statisticienne de l’Unité de soutien
méthodes en Biostatistique et Epidémiologie de l’ULG. Toutes ces personnes ont participé et
rendu possible l’analyse de toutes ces photos de zebrafish aussi bien qualitativement que
quantitativement.
Je voudrais dire un tout grand et énorme merci à tous les gens extérieur au labo qui m’ont
soutenu, évidemment je veux parler de mes ami(e)s et mes parents.
Je commencerais par mes ami(e)s ; Marie-Line, Bernard, Valérie, Geoffroy, Tsvetie, Christel,
Nicole et tous les autres que je n’ai pas cité. Tous ont su être à l’écoute, présent, réconfortant
et motivant pour que je puisse aboutir à la fin de ce travail.
Une pensée avec un grand merci à ma marraine et ses filles qui ont toujours eu des pensées
positives pour moi, même de loin.
Et finalement, le plus grand merci est destiné tout naturellement à mes parents pour m’avoir
soutenue, re- soutenue et re re-soutenue jusqu’au bout. Cela n’a pas été rose tous les jours…
Malgré cela, j’ai pu compter sur leur compréhension et présence. Ils ont toujours respecté mes
choix et même en période de doutes, ils m’ont toujours encouragée à ne pas perdre mes
objectifs de vue.
Table of contents 1

TABLE OF CONTENTS

Abbreviation list...............................................................................................4
Introduction.....................................................................................................6
Foreword......................................................................................................... 6
1. Microgravity and its effects on humans in space............................................................ 7
2. Skeleton development in vertebrates.............................................................................. 9
2.1. Cartilage................................................................................................................... 9
2.1.1 Cartilage development................................................................................. 9
2.1.2 Different cartilage type............................................................................... 10
2.2. Bone and its development.......................................................................................11
2.2.1. Endochondral ossification...........................................................................12
2.2.2. Intramembranous ossification.....................................................................13
2.1. Bone remodeling......................................................................................................... 14
2.3.1 Osteoblasts formation and development into osteocytes........................... 14
2.3.1.1 Some important ossification genes................................................ 15
2.3.2 Osteoclasts................................................................................................. 21
2.3.3 Bone turnover............................................................................................ 22
2.2. Systemic bone regulation........................................................................................... 24
2.2.1. Parathyroid hormone................................................................................. 24
2.2.2. Vitamin D3................................................................................................ 25
2.2.3. Calcium homeostasis by parathyroid hormone and vitamin D3............... 26
2.3. Osteoporosis.............................................................................................................. 27
2.4. Astronaut’s osteoporosis............................................................................................ 30
3. Microgravity and hypergravity simulation experiments.............................................. 33
3.1. Clinostat..................................................................................................................... 33
3.2. Random Positioning Machine.................................................................................... 35
3.3. Rotating Wall Vessel.................................................................................................. 36
3.4. Large Diameter Centrifuge......................................................................................... 36
4. Zebrafish advantages and use as model for genomics analysis.................................... 37
4.1. Genome...................................................................................................................... 37
4.2. Duplication and Conservation.................................................................................... 37
5. Zebrafish as bone model............................................................................................... 38
5.1. General structure ....................................................................................................... 38
5.2. Cartilage development................................................................................................ 40
5.3. Bone development...................................................................................................... 41
5.4. PTH, VitD3 and calcium homeostasis....................................................................... 44
6. Zebrafish as model to study effect of altered gravity................................................... 46
7. Principal objectives of this study.................................................................................. 47
Table of contents 2

Results........................................................................................................... 49
Chapter 1: Head skeletal development and gene expression modulation upon drug
treatments.
1. Effects of drug treatments on head skeletal formation.................................................. 49
2. Modification of gene expression upon drug treatment.................................................. 58
3. Whole genome analysis of gene expression modulation by drugs................................ 60
4. Conclusions................................................................................................................... 67
Chapter 1bis: Function of the Sox4 transcription factors in skeletal development.
1. Sox4 genes in mice and zebrafish................................................................................. 70
2. Expression pattern of sox4a and sox4b......................................................................... 70
3. Expression of sox4a and sox4b during development and upon PTH and
VitD3treatment.......................................................................................................................72
4. Conclusions................................................................................................................... 72
Chapter 2: Head skeletal development and gene expression modulation upon microgravity
simulators.
1. Head skeletal development and gene expression modulation upon microgravity
simulators..................................................................................................................... 74
2. Effects of simulated microgravity on cartilage and bone formation............................. 74
3. Effects of simulated microgravity on stress in larvae................................................... 80
4. Rotating Wall Vessel.................................................................................................... 85
5. Effects of simulated microgravity on gene expression................................................. 87
6. Conclusions................................................................................................................... 97
Chapter 3: Head skeletal development and gene expression modulation upon
hypergravity.
1. Effects of hypergravity on bone and general development......................................... 101
2. Effects of "relative microgravity" on bone and general development........................ 108
2.1. A central gene network is rapidly activated in reduced gravity................................ 112
3. Conclusions................................................................................................................. 118
Discussion................................................................................................... 122
Material and Methods................................................................................ 135
1. Animals procedures..................................................................................................... 135
2. Hormone treatments.................................................................................................... 135
3. Microgravity simulation experiments......................................................................... 136
3.1. Clinostat.................................................................................................................... 136
3.2. Random Positioning Machine................................................................................... 136
3.3. Rotating Wall Vessel................................................................................................ 136
4. Hypergravity experiments in the Large Diameter Centrifuge..................................... 137
5. Stress experiment........................................................................................................ 137
5.1. Dexamethasone......................................................................................................... 137
5.2. Cortisol stress response............................................................................................. 137
5.3. Cortisol measurement............................................................................................... 138
6. Staining methods........................................................................................................ 138
7. Image analysis............................................................................................................ 139
8. Single and fluorescent double whole-mount in situ hybridization............................. 141
Table of contents 3

9. RNA extraction and reverse transcription.................................................................. 141
10. Real Time-PCR......................................................................................................... 142
11. Microarrays expression experiments......................................................................... 142
12. Ingenuity Pathway Analysis...................................................................................... 144
Annexes....................................................................................................... 145
Annex 1: List of primers..................................................................................................145
Annex 2: Morphometric analysis of cartilage staining after 5 days chemical
treatments..........................................................................................................................146
Annex 3: Morphometric analysis of bone staining after 5 days chemical
treatments..........................................................................................................................147
Annex 4: VitD3 microarrays by entrez gene name..........................................................148
Annex 5: PTH microarrays by entrez gene name............................................................ 158
Annex 6: VitD3 and PTH Heat map................................................................................162
Annex 7: Morphometric analysis of cartilage staining after 5 days microgravity simulators
.......................................................................................................................................... 166
Annex 8: Morphometric analysis of bone staining after 5 days microgravity
simulators..........................................................................................................................167
Annex 9: Morphometric analysis of bone staining after 5 days dexamethasone
treatment............................................................................................................................168
Annex 10: Recapitulative table of statistical analysis of RWV in bone evolution image
analysis..............................................................................................................................169
Annex 11: CLINO microarrays by entrez gene name......................................................170
Annex 12: RPM microarrays by entrez gene name..........................................................171
Annex 13: RWV microarrays by entrez gene name.........................................................173
Annex 14: Microgravity microarrays (CLINO, RPM, RWV) by category.....................177
Annex 15: Microgravity (CLINO, RPM, RWV) Heat map.............................................178
Annex 16: Morphometric analysis of bone staining after 5 days relative
microgravity..................................................................................................................... 181
Annex 17: Hypergravity microarrays by entrez gene name............................................ 182
Annex 18: Relative microgravity microarrays (1g) by entrez gene name...................... 186
Annex 19: Relative microgravity microarrays (3g>axe) by entrez gene name.............. 194
Annex 20: Relative microgravity microarrays (3g>1g) by entrez gene name................ 197
Annex 21: Relative microgravity microarrays (1g, 3g>axe, 3g>1g) by category.......... 209
Annex 22: Relative microgravity microarrays (1g, 3g>axe, 3g>1g) Heat map by
category............................................................................................................................ 210
Annex 23: Relative microgravity microarrays (1g, 3g>axe, 3g>1g) Heat map by diseases
and functions.................................................................................................................... 212
Bibliography................................................................................................218
Publications................................................................................................ 243
Abbreviations list 4

Abbreviation list
µg: microgravity CYP24a1: Cytochrome P450, family 24,
subfamily A, and polypeptide 1
µScm-1: microsiemen per centimeter

D
1α(OH)ase: 1,25-dihydroxyvitamin D-1α-
hydrolase DKK1: Dickkopf factors
1,25(OH) D3:1,25- DMSO: dimethylsulfoxid
2
dihydroxycholecalciferol
DNA: deoxyribonucleic acid
11βHSD2: 11β-hydroxysteroid
DPD: deoxypyridinoline
dehydrogenase type 2
dpf: day post-fertilization
25-OH-D: 25-hydroxycholecalciferol
DVL: Disheveled
A
ACTH: adrenocorticotropic hormone E
ECM: extracellular matrix
ALP: Alkaline phosphatase
ERK: extracellular signal regulated kinase
ARED: Advanced Resistive Exercise

Device
ESA: European space agency
B
F
Bglap: bone gla protein or osteocalcin
Fz: Frizzeld
BMD: bone mineral density
G

BMU: Bone multicellular unit g: gravity
Bsp: bone sialoprotein GADD45B: growth arrest and DNA
damage-inducible
C
gag: glycoaminoglycan
CEVIS: cycle ergometer with vibration
isolation and stabilization
gapdh: glyceraldehyde-3-phosphate
dehydrogenase
cNCC: cranial neural crest cells
gfp: green fluorescent protein
Col: collagen
GR: glucocorticoid receptor
CLINO: clinorotation
GSK3β: glycogen synthetase kinase 3-β
CT: calcitonin
H
CTR: Calcitonin receptor
HES: Hairy enhancer of split
CRF: corticotropin-releasing factor
Hg: hypergravity
CRLR: Calcitonin receptor-like receptor
hpf: hour post-fertilization
CTX: C-telopeptide
HPI : hypothalamus-pituitary-interrenal
Abbreviations list 5

I PTH: parathyroid hormone
iRED: interim resistive exercise device
PTHR1: parathyroid hormone receptor
type1
Ihh: Indian Hedgehog
PTHrP: parathyroid hormone-related
IPA: Ingenuity Pathway Analysis
protein
ISH: in situ hybridization

PYD: pyridinium or pyridinoline
ISS: International Space Station
R
J
RANK: receptor activator of nuclear factor
JNK: c-Jun terminal kinase κB
L RANKL: receptor activator of nuclear
factor κB ligand
LDC: large diameter centrifuge
RHCG: Rh Type C glycoprotein gene
Lrp 5/6: low density lipoprotein receptor-
related protein 5 or 6
RNA: ribonucleic acid
M
RPM: random positioning machine
MCR2: melanocortin 2 receptor

rpm: rotation per minute
MITF: microphthalmia transcription factor
RT-PCR: reverse transcriptase –
Mo: Morpholino polymerase chain reaction
MR: mineralocorticoid receptor RWV: rotating wall vessel
mRNA: messenger ribonucleic acid S
SAS: space adaptation syndrome
N
NFATc1: nuclear factor activated T cells, SMS: space motion sickness
cytoplasmic, calcineurin-dependent 1
Spp1: osteopontin
NCC: neural crest cells
STC: stanniocalcin
NDRG: new family of differentiation-
related genes T
TNF: tumor necrosis factor
NDRG2: N-myc downstream regulated
gene 2 TRAP: tartrate-resistant acid phosphatase
NTX: N-telopepetide TTN: titine
O TVIS: vibration isolation and stabilization
OPG: osteoprotegrin
U
OPPG: osteoporosis pseudoglioma UBB: ultimobranchial bodies
Osx: osterix V

VitD: Vitamin D
P
PPARγ2: proliferator activated receptor γ2 VDR: Vitamin D receptor
Introduction
Introduction 6

Foreword
Human has always been fascinated the mystery of space. This space conquest has begun in
1957 with the first Russian spaceflight, Sputnik 1. Then, Americans have reached the moon
and walked on it in June 1969. Since then, several Gemini missions and others have followed.
Human’s ambitions did not stop with the construction of the International Space Station (ISS),
where the mission duration extends up to 6 months or longer. The ambition still progresses
with the aim of Mars exploration in 2030. However, these space explorations are not without
consequences (Hughes-Fulford 2011, McCarthy 2011).
Since the very beginning of spaceflight, with short-term missions, the effects of microgravity
on the astronauts became apparent, inducing several physiological problems such as
vestibular troubles, loss of muscular mass, circulatory problems, as well as weariness and
psychological troubles (Blaber and all, 2010; Mc Carty,2011; Fong, 2008). Unfortunately,
these effects do not stop there. With long duration spaceflight from 1 to 6 month, the
astronauts presented a bone loss of 1 to 2% per month, due to a decrease of bone formation
and a normal or increased rate of bone resorption (Collet, Uebelhart et al. 1997, Carmeliet and
Bouillon 1999). This important bone loss is called the "astronaut's osteoporosis".
The mechanisms involved in this microgravity effect on bone are unknown despite many
studies attempting to explain the process. Even if the astronauts are submitted to extensive
physical exercise before and during the flight, bone loss remains significant and the astronauts
do not recover their previous total bone mass after a recovery period as long as the
spaceflight. Therefore, the fracture risk is increased on ground after return (Collet, Uebelhart
et al. 1997, Vico, Collet et al. 2000, Sibonga 2013).
Thus, if humanity want's to achieve its ambition of a manned spaceflight to Mars (where the
flight duration to reach Mars takes at least 6 months, spend some time for exploration and 6
months to return to Earth), it is imperative to find solutions, among others, to prevent bone
loss.
Currently, the research uses various models and methods for a better comprehension of the
process of bone loss, such as bed-rest, cultured cells, hindlimb unloading (suspension by the
tail) rats or fish. The present work integrates into the bone problematic using the zebrafish as
a model to study cartilage and bone formation under different environmental conditions, such
as drug treatment, microgravity simulation or hypergravity.
Introduction 7

1. Microgravity and its effects on humans in space
The gravitational force is a vector composed of a magnitude and a direction facing the center
of the earth. Isaac Newton has discovered the universal law of gravitation in 1665-1666 and
has developed the formula to evaluate the attractive force between two bodies:
F = G Mm/d²
g u

with “M” the mass of earth, “m” the mass of the object, “d” the distance between the center of
the two masses and “G ” the universal gravitational constant = 6.67 x 10-11 m³/kg.s². The
u
higher the distance between the two bodies, the smaller the gravitational force between them.
Any object on the earth's surface is submitted to a gravity force equal to 1g. The microgravity
experienced during a space mission in orbit is not due to the distance between the spacecraft
and earth, rather the gravitational force is decreased by the centrifugal force produced by the
speed of the spacecraft in a circular orbit, actually initiating a continuous free fall (Fig. 1).
Taking into consideration the mass and the gravitational force, the microgravity corresponds
to 10-3g on ISS, up to 10-5g in the drop towers and 10-2g during parabolic flight. The zero
gravity (0g) is actually never reached (Morey-Holton 2003, Schmidt 2004).

Figure 1: Forces acting on the spacecraft's trajectory leading to free fall and circling of
the earth (Morey-Holton 2003).

Astronauts are also submitted to microgravity and experience several physiological effects
during each flight. The human body is designed to live in 1g, not in microgravity. Here, we
describe the most affected systems. The cardiovascular system endures some modifications.
The fluids (blood and plasma) are shifted from the lower to the upper part of the body,
inducing a face oedema during the first days in space. Higher blood volume in the head