The Transcriptome Of The Suprachiasmatic Nucleus And Its Response To Photic Stimuli
A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy
By
Veronica Marie Porterfield
December, 2008
Dissertation written by Veronica M. Porterfield
B.S. Westminster College, 2002 Ph.D. Kent State University, 2008
Approved by
____Dr. Eric M. Mintz______, Chair, Doctoral Dissertation Committee
____Dr. Gail C. Fraizer______, Member, Doctoral Dissertation Committee
____Dr. Jennifer L. Marcinkiewicz __, Member, Doctoral Dissertation Committee
____Dr. James L. Blank______, Member, Doctoral Dissertation Committee
____Dr. J. David Glass______, Member, Doctoral Dissertation Committee
Accepted by
_____Dr. Robert V. Dorman______, Director, School of Biomedical Sciences
_____Dr. John R. D. Stalvey______, Dean, College of Arts and Sciences
ii Table of Contents
Page
List of Figures……………………………………………………………..………...…viii
List of Tables………………………………………………………………………….....x
List of Abbreviations……………………………………………………………………xii
Acknowledgements………………………………………………………………….…xv
Chapter I. Introduction…………………………………………………….……………1
General Background……………………………………………………………1
The Suprachiasmatic Nucleus………………………………..………………..4
Molecular Biology of the Clock………………………………………………...7
Effect of Photic Stimulation on the Molecular Clock……….………………..9
Immediate Early Genes………………………...……………………………..11
Specific Aims……………………………..…………………………………….12
Chapter II. Identification of light-induced genes in the hamster suprachiasmatic nucleus………………………………..………………………………………………...14
Introduction……………………..………………………………………………14
Methods………………………….……………………………………………..15
Animal Handling and Tissue Prep…………………..……………………….15
iii
Microarrays……………………………..………………………………………16
Primer Design……………………………………………….…………………16
Statistical Analysis for Microarrays………………………..………………...17
Method 1…………………………………………..……………17
Method 2a…………………………………..…………………..18
Method 2b…………………………………..…………………..18
Results …………………………...………………………………………18
Discussion…………….………………………………………………………..27
Chapter III. Identification of novel light-induced genes in the suprachiasmatic nucleus……………………..…………………………………………………………...31
Abstract……………..…………………………………………………………..31
Background……………….…………………………….……………...31
Results…………………………….……………………………………31
Conclusions…………………………………………….………………32
Background……………………………………….……………………………32
Methods………………………………………….……………………………..34
Animals……………………………………………..…………………..34
Laser Capture Microscopy/RNA purification………………..………35
Microarrays…………………………………………………..…………36
Real time PCR……………………………………….………………37
iv
Analysis of evolutionary conservation of CRE and TATA elments
in the promoter sequences……………………………………………38
Results…………………………………………………………………...... 39
Light-induced Immediate Early genes in the SCN…………………39
Analysis of Promoter Regions of light-induced genes……………..43
Discussion………………………………….…………………………………..50
Conclusions……………………………….……………………………………55
Chapter IV. Time course of light-induced genes…………………………………...56
Introduction………….………………………………………………………....56
Methods and Materials…………………………………...…………………...58
Animals…………………………..……………………………………..58
Laser Capture Microscopy/RNA purification……..…………………58
Real Time PCR………………………….……………………………..59
Results ……………………...……………………………………………60
Discussion……….……………………………………………………………..64
Chapter V. Localization of Rrad mRNA expression in the suprachiasmatic nucleus………………………………………………………………………………….67
Introduction………..……………………………………………………………67
Methods and Materials……………………...………………………………...70
Animals………………………………..………………………………..70
v
Riboprobe synthesis…………………………...……………………...70
Tissue prep and in situ hybridization…………………..…………….75
5’ RACE…………………………..…………………………………….76
Results…………………….……………………………………………………77
Discussion……………………….……………………………………………..87
Chapter VI. Meta-analysis of the neurotransmitter and neuropeptide systems of the suprachiasmatic nucleus………………………………...……………………….89
Introduction…………………..…………………………………………………89
Methods……………………….………………………………………………..91
Results………………………….………………………………………………92
Comparison of genes present on each microarray…………..…….92
Classical Neurotransmitters…………..……………………………...94
Neuropeptides and Other Notable Genes………………...……….102
Overlap in expression between studies……………………………107
Discussion………………………….…………………………………………110
Chapter VII. Global Discussion………………...…………………………………...116
Future Directions……………….…………………………………………….120
Final Thoughts……………….……………………………………………….121
vi
Appendix…………………..…………………………………………………………..123
Supplementary Notes for Table 3-2…………….………………………….123
Reference List…………….…………………………………………………………..124
vii
List of Figures
Page
Chapter I. Introduction
Fig. 1-1. Phase Response Curve…………….………………………………………..3
Fig. 1-2. Suprachiasmatic Nucleus………..…………………………………………..6
Fig. 1-3. Schematic representation of the circadian molecular clock….………..…8
Chapter II. Identification of light-induced genes in the hamster suprachiasmatic nucleus
Fig. 2-1. Laser capture microscopy…….……………………………………………19
Fig. 2-2. Bioanalyzer verification of RNA quality…………...………………………20
Fig. 2-3. Hybridization……….………………………………………………………...21
Fig. 2-4. Bioanalyzer verification of qPCR product sizes………………………….25
Fig. 2-5. Fos and Egr1 condition trees……………..………………………………..26
Fig. 2-6. Plot of genes…………………………………………………………………28
Chapter III. Identification of novel light-induced genes in the suprachiasmatic.
Fig. 3-1. Laser capture microscopy and gene expression in a response to a light
pulse………..…………………………………………………………………...40
Fig. 3-2. Gene expression after light pulse vs. sham light pulse………….……...42
Fig. 3-3. Fold change of light-induced genes…………….…………………………44
viii
Fig. 3-4. Conserved promoter elements in light-induced genes…………….……47
Chapter IV. Time course of light-induced genes
Fig. 4-1. Genes that represent the first pattern of expression……….……………61
Fig. 4-2. Per2 representation of the second pattern of expression…...………….62
Fig. 4-3. Genes that represent the third pattern of expression…………………...63
Chapter V. Localization of Rrad mRNA expression in the suprachiasmatic nucleus
Fig. 5-1. Rrad sequence and probe locations……………...……………………….73
Fig. 5-2. Probe 1 hybridization in heart………………..…………………………….78
Fig. 5-3. Probe 1 hybridization in the SCN………………..………………………...79
Fig. 5-4. Rrad individual probes on Affymetrix microarrays………….……………80
Fig. 5-5. Probe 3 hybridization in heart……………………..……………………….82
Fig. 5-6. Probe 4 hybridization in heart………………………..…………………….83
Fig. 5-7. Rrad hybridization from Allen Brain Atlas……………………..………….85
Chapter VI. Meta-analysis of the neurotransmitter and neuropeptide systems of the suprachiasmatic nucleus
Fig. 6-1. Overlap in neurotransmitter system expression……..…………………108
Fig. 6-2. Overlap in neuropeptide system expression……..……………………..109
ix
List of Tables
Page
Chapter II. Identification of light-induced genes in the hamster suprachiasmatic nucleus.
Table 2-1. Up and down-regulated genes following a light pulse……….……..…23
Table 2-2. Fold change of genes…………………….………………………………24
Table 2-3. Fold change of additional genes………………………………………...29
Chapter III. Identification of novel light-induced genes in the suprachiasmatic.
Table 3-1. Fold change in expression following a light pulse……………..……...45
Table 3-2. Conservation of CRE and TATA elements in light induced genes…..48
Table 3-3. CRE and TATA elements in selected clock genes…….……………...49
Chapter V. Localization of Rrad mRNA expression in the suprachiasmatic nucleus.
Table 5-1. Riboprobe sequences………….…………………………………………72
Chapter VI. Meta-analysis of the neurotransmitter and neuropeptide systems of the suprachiasmatic nucleus
Table 6-1. Comparison of genes present on each microarray……………………93
Table 6-2. GABA………….…………………………………………………………...95
x
Table 6-3. Glutamate………………………………………………………………….97
Table 6-4. Acetylcholine and Dopamine……………..……………………………...98
Table 6-5. Histamine and Serotonin……………..…………………………………100
Table 6-6. Norepinephrine, Epinephrine, and Glycine………...…………………101
Table 6-7. AVP, GRP, NPY, PACAP, and VIP………………..…………………..103
Table 6-8. GnRH, Hyprocretin, Leptin and Melatonin……………………………104
Table 6-9. Calbindin, Neurotensin, Oxytocin, Preproenkephalin, Prokineticin2,
Somatostatin, and Tachykinin………………………..……………………………..106
xi
List of Abbreviations
5'RACE- Rapid amplification of cDNA ends
AMPA- alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
AVP- Arginine vasopressin
Bmal1- brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)- like
Btg2- B cell translocation gene 2
CalB- calbindin
Cdh2- cadherin 2 cebpb- CCAAT enhancer binding protein beta
Chat- Choline acetyltransferase
Clock- Circadian locomotor output cycles kaput
CRE- cAMP response element
CREB- cAMP response element binding
Cry1- cryptochrome 1
Cry2- cryptocrhome 2
CT- circadian time
DD- constant dark
Dusp1- dual specificity phosphatase 1
Egr1- early growth response 1
Egr2- early growth response 2
xii
Egr3- early growth response 3
ERK- extracellular regulated kinase
Fos- FBJ osteosarcoma oncogen
GABA- Gamma-aminobutyric acid
Gad1- glutamic acid decarboxylase 1
Gadd45b- growth arrest and DNA-damage-inducible 45 beta
Gapdh- Glyceraldehyde 3-phosphate dehydrogenase
GC-RMA- GC robust multi-array analysis
GEO- Gene Expression Omnibus
GHT- geniculohypothalamic tract
GnRH- gonadotropin releasing hormone
GRP- gastrin releasing peptide
Hipk3- homeodomain interacting protein kinase 3
Jun- Jun oncogene
Klf4- kruppel like factor 4
LCM- laser capture microscopy
LD- light dark cycle
MAPK- mitogen-activated protein kinase
MEK1/2- Map kinase kinase or Erk kinase 1/2
Mkp1- Map kinase phoshpatase 1
NMDA- N-methyl-D-aspartic acid
NPY- neuropeptide Y
xiii
Nr4a1- nuclear receptor subfamily 4, group A, member 1
Nrg3- neuregulin 3
Oprl1- opioid receptor like 1
Oprlm1- opioid receptor, mu 1
PACAP- pituitary adenylate cyclase activating polypeptide
PCR- polymerase chain reaction
Per1- period 1
Per2- period 2
Pim3- proviral integration 3
Ppp2r5c- protein phosphatase 2, regulatory subunit B(B56), gamma isoform qPCR- quantitative polymerase chain reaction qRTPCR- quantitative real time polymerase chain reaction
RHT- retinal hypothalamic tract
Rrad- Ras-related associated with diabetes
RT-PCR- real time polymerase chain reaction
SCN- suprachiasmatic nucleus
Tiparp- TCDD-inducible poly(ADP-ribose) polymerase
Ube2i- ubiquitin-conjugating enzyme E2i
VIP- vasoactive intestingal polypeptide
xiv
Acknowledgements
First I would like to thank my advisor, Dr. Eric Mintz, for his support and
guidance over the years. You gave me a lab to work in, when I had no lab. I
have learned a lot over the years from you, seeing that I am not a neuroscientist,
and I hope that some of my molecular biology knowledge has rubbed off on you.
I thank you for the opportunities that have come my way from working in you lab and I wish you continued success.
To my committee members, Dr. Gail Fraizer, Dr. Jennifer Marcinkiewicz,
Dr. James Blank, and Dr. J. David Glass, thank you for your help and guidance
over the years. A special thanks to Drs. Fraizer and Marcinkiewicz for their help
when I had a molecular question. I would like to thank Dr. Helen Piontkivska for
her help with the bioinformatics from my first publication. To Dr. Heather
Caldwell, thank you so much for all of your help with in situ hybridization even
though Rrad got the better of us!
To my labmates over the years, thank you for all of the fun times we had
in and out of the lab. A special thanks to George Kallingal and Erin Gilbert, you
guys were there over the years, to either bounce ideas off of (whether it was for a
project or a random game) and just to talk when times got stressful. I especially
would like to thank Erin Herrman, for always being there for me no matter what
xv the situation was. We have definitely gone through a lot over the last 5 years! I wish all of you the best of luck in your future endeavors!
Last but definitely not least, I would like to thank my family. To my brother, Russell Jr., thank you for being you and all of your help over the years.
To my wonderful parents, Marlene and Russell, I can never thank you enough for all of the help and support you guys have given me over the years. Thank you for helping move just about every year that I was out here in Kent, and for all of your help with Jake. Most importantly, thank you for encouraging me ever since I can remember to go to school and become all that I can be, I would have never been able to come this far without you.
xvi
Chapter I
Introduction
General Background
Even before the invention of the modern clock, humans, mammals, plants and even bacteria had developed an innate way to keep time. The Earth’s rotation provided the hour hands of the clock, enabling its occupants to establish the proper timing of their day to day needs to approximately 24 hours. Webster’s dictionary defines timing as the selection for maximum effect of the precise moment for beginning or doing something. By adapting to one’s environment, organisms can essentially maximize their daily activities or timing to ensure their survival. For example, nocturnal animals are most active during the night and by doing so avoid diurnal predators that are active during the day. For the early evolution of biological clocks Pittendrigh (1993) suggested the “escape from light/UV” hypothesis. This explains the early evolution of clocks in prokaryotes and eukaryotes because before the ozone layer was in place they were highly susceptible to UV-radiation damage. To minimize DNA damage by UV radiation, the S phase of the cell cycle was restricted to night (Rosato and Kyriacou, 2002;
Pittendrigh, 1993). This then helped to evolve biological clocks as we know them today; internal clocks synchronized to the external world that allows an organism to predict and prepare for daily environmental changes (Pittendrigh, 1993).
1 2
Circadian rhythms fall under the control of circadian clock. There are
three criteria that classify these rhythms as circadian. These include: (1) the
overt circadian rhythm persists in constant temperature and constant light or
constant dark conditions with a period of approximately 24 hours, (2) the rhythms
are temperature compensated, and (3) these rhythms of approximately 24 hours can be entrained by certain 24 hour environmental cues (i.e. light-dark cycles, temperature cycles, or other stimuli) (Dunlap et al., 2004). These rhythms organize the physiology and behavior of the organism such that the various processes necessary to life are scheduled to occur at the most appropriate time of day or night. Since the clock is not exactly 24 hours, it must rely on various environmental stimuli to entrain and reset itself to a 24 hour day. An important cue for entrainment is light, which enables an organism to anticipate day length, thus allowing it to anticipate environmental events (Pittendrigh and Daan, 1976).
The timing of the clock can be shifted by both photic (i.e. light) and non-photic
(i.e. temperature, drugs) stimuli. How light affects the clock is determined by the phase response curve (PRC). The PRC shows that light presented in the early night delays the phase of the clock, light during the late night advances the clock, and light presented during the subjective day has little or no effect on clock phase (Pittendrigh et al., 1976) (Figure 1-1).
3
Figure 1-1 The phase response curve shows how light exposure at a given times, effects the behavior of the animal. The example shown is representative for hamsters. Light given during the subjective day has little to no effect on shifting the clock, and is termed “Dead Zone”. Light presented during the early subjective night causes a delay, and light presented during the late night causes an advance. The white area on the X-axis designates day, and the black shaded area represents night. 4
The Suprachiasmatic Nucleus
The circadian clock is located in an area of the brain called the
suprachiasmatic nucleus (SCN). The SCN lies directly above the optic chiasm
and is lateral to the third ventricle in the anterior hypothalamus. It is a structure
of 2 paired nuclei that is compromised of approximately 20,000 neurons (10,000
each nucleus) (Inouye and Kawamura, 1979). The SCN was discovered to bethe
master pacemaker through hypothalamic lesion studies, where removal of the
SCN abolished circadian rhythmicity (Stephan and Zucker, 1972; Moore and
Eichler, 1972). To further show that the SCN was the master clock, these
rhythms could be restored with SCN transplants (Lehman et al., 1987).
However, the restored rhythms are not that of the host but that of the donor SCN
(Ralph et al., 1990; Sujino et al., 2003). These experiments show that the SCN is important for the generation of circadian rhythms.
There are three main inputs into the SCN: the retinal hypothalamic tract
(RHT), the geniculohypothalamic tract (GHT), and the raphe nucleus. The most
important of these 3 for light entrainment is the RHT, which directly connects the
retina to the SCN (Moore and Lenn, 1972; Hendrickson, Wagoner, and Cowan,
1972). The primary neurotransmitter for this pathway is glutamate (Castel et al.,
1993b; Mintz and Albers, 1997; Mintz, Marvel, Gillespie, Price, and Albers, 1999;
Ebling, 1996a). There are other neurotransmitters that are localized in RHT
terminals in addition to glutamate and these include aspartate, substance P, and
PACAP (Castel et al., 1993a; Ebling, 1996b). The RHT was determined to be 5
essential for photic entrainment because surgical destruction of this projection
leads to the inability to entrain to environmental light-dark cycles (Johnson et al.,
1988). The GHT connects the intergeniculate leaflet (IGL) of the hypothalamus
to the SCN (Ribak and Peters, 1975), and the primary neurotransmitter is
neuropeptide Y (NPY) (Card and Moore, 1982). The third input to the SCN is the
raphe nucleus which utilizes serotonin as its neurotransmitter (Morin and Meyer-
Bernstein, 1999). The GHT and raphe nucleus are not necessary for photic
entrainment (Meyer-Bernstein et al., 1997; Harrington and Rusak, 1986), and
with destruction of the RHT leading the inability to entrain to the environment,
these data suggest that the RHT is the main input into the SCN that conveys
information about the environment enabling entrainment to the light-dark cycles.
The composition of the SCN is not a homogenous population of neurons.
It contains heterogenous populations of neurons that define different regions of
the SCN based on the neurotransmitters and neuromodulators in each region.
The SCN is most classically viewed as being divided into the dorsomedial shell and the ventrolateral core (Moore, 1996) (Figure 1-2). The shell of the SCN is
the rhythmic portion of the SCN and it expresses arginine-vasopression (AVP)
(Card and Moore, 1984b; Abrahamson and Moore, 2001a; Moore et al., 2002a).
The core is the light-inducible region of the SCN and it expresses vasoactive
intestinal polypeptide (VIP) and gastrin-releasing peptide (GRP) immunoreactive
cells (Card and Moore, 1984a; Tanaka et al., 1997c; Abrahamson and Moore,
2001b; Moore et al., 2002b; Karatsoreos, Yan et al., 2004; Morin et al., 2006). 6
Figure 1-2 Suprachiasmatic Nucleus Schematic representation of the SCN and localization of neurotransmitters and neuropeptides within it. For ease of drawing, the hamster SCN is represented here. The light blue represents cells immunoreactive for VIP, red represents calbindin, dark blue represents GRP, and purple represents the Cap, which compromise the core of the SCN. The yellow represents AVP and makes up the shell. Adapted from Antle and Silver, 2005. 7
However, this view of the SCN is a simplified version, and there are even more subdivisions within the core. For example, in hamsters there is a calbindin
(CalB) immunoreactive cluster (Silver et al., 1996). Mice and rats also have
CalB, but it is not in a well defined cluster as it is in hamsters. In this dissertation,
I attempt to compile of list of these and other neurotransmitters found in the SCN
based on meta-analysis of microarray data.
Molecular Biology of the Circadian Clock
The basic molecular mechanism of the mammalian circadian clock is now
fairly well defined and several reviews have been written on the subject (Hastings
and Herzog, 2004b; Okamura, 2004; Zhang et al., 2004). At the core of the clock
is a feedback loop in which cyclically expressed clock gene products negatively
regulate their own expression in a period of approximately 24 hours (Hastings,
1997; Whitmore et al., 1998; Dunlap, 1999b). These genes include the three
Period genes (Per1, Per2, and Per3) and the two Cryptochrome genes (Cry1 and
Cry2). Along with the negative regulators, there are two transcriptional activator
genes, Clock and Bmal1 (Dunlap, 1999a; Young, 2000; Shearman et al., 2000)
that activate the transcription of the Per and Cry genes. In addition, Bmal1 is
also regulated by Reverbα, which suppresses Bmal1, and this forms the positive feedback loop.
During the day, transcriptional activation of Per1, Per2, Cry1I, and Cry2,
occurs by CLOCK and BMAL1 forming heterodimers that bind to E-boxes in their
promoters (Figure 1-3). PER and CRY accumulate in the cytoplasm where they 8
Figure 1-3 Schematic representation of the circadian molecular clock. CLOCK and BMAL1 form heterodimers in the cytoplasm and translocate to the nucleus to activate the period, and cryptochrome genes along with Rev-erbα by binding to their E-box promoter. PER is phosphorylated by casein kinase 1ε and forms a heterodimer with CRY. They translocate back to the nucleus where they act to inhibit their own transcription by inhibiting CLOCK/BMAL1 transcription of their gene. This forms the negative feedback portion of the loop. Activation of Rev-erbα leads to inhibition of Bmal1, that inhibition is released once PER/CRY inhibit CLOCK/BMAL activation of their transcription factors. This provides the positive portion of the feed back loop. The lines with arrows at the end represent activation, where as lines with a vertical line at the end indicate inhibition. The pink star represents phosphorylation via casein kinase1ε. Adapted from Hardin 2004. 9
form PER/CRY heterodimers, and then translocate into the nucleus (Kume et al.,
1999a). Here they interact with ClOCK/BMAL1 which ultimately inhibits Per and
Cry transcription (Gekakis et al., 1998; Kume et al., 1999b). Not only does
CLOCK/BMAL1 activate the Per and Cry genes, it also activates Reverbα which
inhibits Bmal1 transcription (Preitner et al., 2002b; Ueda et al., 2002b). This
inhibition is released with the disappearance of REVERBα via the same
PER/CRY heterodimers that in addition to inhibiting their own transcription, inhibit
the transcription of Reverbα leading to Bmal1 transcription (Preitner et al., 2002a;
Ueda et al., 2002a). This allows peak expression of Bmal1 to occur at the end of the circadian night (Hastings and Herzog, 2004c) in time to start transcriptional activation of Per and Cry again. Thus the positive and negative portions of the feedback loop are in antiphase with each other providing the stability and maintenance of the oscillation of gene and protein products to a period of about
24 hours (Hastings and Herzog, 2004a).
Effect of Photic Stimulation on the Molecular Clock
As mentioned earlier, depending on time of day photic stimulation is given
determines the effect it has on the circadian clock. Light during the early
subjective night will cause a phase delay and light during the late subjective night
will produce a phase advance. Per1, along with being rhythmically expressed is
also induced by light (Kornhauser et al., 1996; Shigeyoshi et al., 1997b). Light is
transmitted from the eyes to the retinorecipient neurons of the SCN via retinal
ganglion cells. Retinal ganglion cell axons innervate the SCN and release 10
glutamate onto the core of the SCN. Glutamate binds to NMDA receptors
causing depolarization of the neurons resulting in an influx of calcium which
activates MAPK leading to CREB phosphorylation (Antle et al., 2005; Obrietan et
al., 1998). Activated CREB binds to CRE elements in Per1 and Per2 promoters
activating their transcription in the core. Per1’s response to light suggests that its
induction is an important event for light induced resetting of the circadian clock
(Shigeyoshi et al., 1997a; Albrecht et al., 1997b). Per2 is also activated by light presented at night, but only at a time when a phase delay occurs (Albrecht et al.,
1997a; Yan and Okamura, 2002a; Yan and Silver, 2004b). Once Per1 and Per2 have been activated in the core, a delaying light pulse will continue the spread of
Per2 to the shell of the SCN and an advancing light pulse will continue the spread of Per1 to the shell (Yan and Okamura, 2002b; Yan and Silver, 2004a).
While there are other signaling pathways that are activated by light during
the subjective night, the MAPK pathway has garnished the most attention. The
MAPK pathway has been found to be very important in the photic induction
signaling pathway, activating immediate early genes (c-fos, egr1, and JunB)
along with CRE. Inhibition of MAPK with the MEK1/2 inhibitor U0126, blocks
expression of the light induced immediate early genes c-fos, egr1 and JunB
(Dziema et al., 2003b). Infusion with U0126 also leads to a reduction in light-
induced phase shifting (Butcher et al., 2002). These suggest that MAPK plays
an important part in the phase shifting induced by light. 11
Immediate early genes
For the purposes of this dissertation we focused our attention on genes
that were rapidly induced by light in the SCN. There are several known light
induced genes in the SCN, and aside from the period genes, their function in the
circadian system is unknown. Some of the best known and well characterized of
these immediate early genes (Fos, Egr1 and Mkp1) will now be discussed. Fos,
a member of the leucine zipper transcription factor family, has rapid induction of its mRNA expression after being presented with stimuli, and its protein is used as a neuronal marker for light-like activity. Behavioral circadian rhythms are still expressed in fos knockout mice as is a phase response curve to light and both are attenuated (Honrado et al., 1996). The threshold for photic induction for c-fos is similar to the threshold for behavioral phase shifts, which suggests a common mechanism (Travnickova et al., 1996) but c-fos can be dissociated from behavioral shifts (Colwell et al., 1993; Schwartz et al., 1996; Edelstein et al.,
2003). This suggests that while Fos is induced by light, it is not necessary for behavioral phase shifts to occur.
Egr1 encodes a zinc finger transcription factor, and is also induced by light
in a phase dependent manner (Rusak et al., 1990; Rusak et al., 1992; Sutin and
Kilduff, 1992; Tanaka et al., 1997a). The expression of egr1 occurs over a
broader area of the SCN than does c-fos, with highest expression in the
ventrolateral section of the SCN and some in the dorsomedial portion (Tanaka et
al., 1997b). Knockout studies with Egr1 in mice show that the knockouts exhibit 12
normal entrainment patterns and respond to light in the same way as their wild-
type counterparts (Kilduff et al., 1998b). Taken together, these data suggest that
egr1 is not an essential component of the pathway for entrainment or in the
phase shifting of locomotor activity in mice (Kilduff et al., 1998a).
A newly discovered light induced gene in the photic signaling pathway is
Dusp1, which encodes Map Kinase Phosphatase 1 (MKP1). Doi et al. (2007) first described Mkp1 as a light induced gene that was also under circadian control. Mkp1 is known to dephosphorylate phospho-mitogen-activated protein kinases (phospho-MAPK) (Charles et al., 1993). Mkp1 is induced by stimulation,
such as serum shock or photic stimuli, and it feeds back to inactivate phospho-
MAPK mediated signal transduction (Camps et al., 2000; Keyse, 2000). One of
the critical steps in the response of the SCN to photic signaling is the
phosphorylation of the extracellular signal regulated kinases (ERK), leading to
phosphorylation of CREB and changes in transcription (Dziema et al., 2003a).
Through inhibition of MAPK, Doi et al. (2007) found a reduction in the induction of
mkp1 showing that mkp1 induction is downstream to MAPK activation, similar to
that of per1 (Doi et al., 2007). This data suggests that while some immediate
early genes appear to have no function in the circadian system, some genes may
function in a circadian manner.
Specific Aims
There are 5 specific aims that will be addressed in this dissertation. The
first aim (Chapter II) focuses on finding light-induced genes in the SCN of 13
hamsters following a light pulse during the early night. The second aim (Chapter
III) also looks at light induced genes following a light pulse during the early night but in the mouse SCN. Aim two also undertakes comparative evolutionary genomic analysis to identify common activation mechanisms among light induced genes. The first two aims utilize laser capture microscopy, microarray analysis and qPCR analysis to find light induced genes for their respective studies. The third aim (Chapter IV) examines the time course of mRNA expression for those genes shown to be upregulated by light in the second aim. The fourth aim
(Chapter V) of this dissertation looks to find the mRNA localization of Rrad in the
SCN with in situ hybridization. The fifth aim (Chapter VI) compares 3 microarray studies done with SCN tissue sample to compile a list of the neurotransmitter/neuropeptide system (enzymes, receptors and transporters) and to see how affective each study was at finding these components, how tissue collection effected the data, and what species differences could be seen.
Overall the data generated from this dissertation will add to our knowledge of light induced genes in the SCN, what roles they may play in photic induction, and generate a comprehensive list of neurotransmitter/neuropeptide systems located in the SCN. Chapter II
Identification of light-induced genes in the hamster suprachiasmatic nucleus
Introduction
Daily cycles of physiology and behavior are driven by endogenous
circadian clocks. When housed in constant darkness (DD), hamsters will still
show a daily rhythm of activity that is close to 24 hours without any external cues
(Pittendrigh and Daan, 1976; Weinert et al., 2001). Normally, however, these rhythms are synchronized on a daily basis by environmental light-dark (LD) cycles. When animals are exposed to light during the early subjective night, their clocks will be shifted such that a phase delay occurs, meaning they will start their daily activity at a later time the following day. A phase advance happens when the light exposure is during late subjective night. The molecular mechanisms underlying these phase shifts are not completely understood.
The master circadian clock is located in the suprachiasmatic nucleus
(SCN) of the hypothalamus (Hastings and Herzog, 2004). Several genes are known to be induced by light in the SCN, including the immediate-early genes c- fos and egr1 (Kornhauser et al., 1990; Rusak et al., 1990; Aronin et al., 1990;
Tanaka et al., 1997; Dziema et al., 2003). In this study, we attempt to perform a
comprehensive screen for light-induced changes in gene expression in Syrian
14 15
hamsters. While hamsters are an excellent species to study for circadian research, one limitation lies in the fact that their genome is not sequenced, and therefore a species-specific microarray is not available. However, it is possible to do cross-species array hybridization and numerous studies have utilized this technique (Chismar et al., 2002; Ji et al., 2004; Shah et al., 2004; Higgins et al.,
2003; Grigoryev et al., 2004). The Affymetrix Mouse 430A 2.0 GeneChips utilizes a set of eleven 25-mer probes for each gene on the array. With cross- species hybridization there is a chance that some of the probes will fail to hybridize due to the fact that the sequences are different and this can increases the number of false negatives. By having 11 different probes for each gene on the array, this increases the possibility of having probes with enough sequence similarity with the target transcript to obtain a decent measure of its expression
(Ji et al., 2004). Secondary goals of this study include validation of statistical approaches to analyzing cross-species hybridization experiments and validation of our approach to real-time PCR analysis of laser-captured SCN tissue.
Methods:
Animal handling and Tissue prep
Adult male Syrian hamsters (Mesocricetus auratus) were bred at Kent
State University from stock purchased from Harlan Sprague-Dawley, Inc.
Hamsters entrained to a 14:10 LD cycle for 2 weeks, then moved to DD where they are allowed to free run for 7 days. At circadian time (CT) 13, hamsters were given either a light pulse (300 lux) or a dark (sham) pulse for a duration of 30 16
minutes. At the end of the treatment, the hamsters were decapitated and their
brains were quickly removed and frozen in cold isopentane. Twelve µm thick sections of the SCN were microdissected using laser capture microscopy, and total RNA was extracted, purified and checked for integrity on an Agilent
Bioanalyzer.
Microarrays
Total RNA was processed through two rounds of linear amplification using
the Riobamp HS amplification kit (Arcturus). The samples were labeled and
hybridized to Affymetrix Mouse 430A 2.0 genechips using the Affymetrix
standard protocol. Genechips were analyzed using GeneSpring software.
Initially, the GC-RMA method was used to generate signal intensities. Only
those genes that showed a 50% increase or decrease in expression and had a
significant T-test for change in expression were considered to have shown a
significant change in expression. Primers were then designed for these genes
for quantitative real time PCR (qRTPCR) analysis, and the experiment was
repeated to generate RNA for the analysis of individual genes.
Primer design
Primers were designed using the Primer Express 2.0 software (Applied
Biosystems, Inc.) for sequences available in hamster and for those not available
in hamsters. However, if no hamster sequence was available, the mouse
sequence was used as the basis for the primer design. Mouse, rat and human
sequences were aligned, and primers were formed from sequence regions of 17
100% identity where possible. This hopefully will provide an area that may also be conserved in hamsters and provide an area to use to validate gene expression.
Statistical analysis for microarrays
Method 1
GC-RMA
Signal values for each probe set were normalized across the twelve genechips to the median value. After filtering out the probe sets with minimal signal intensities, an analysis of variance was performed to detect any genes that showed significant differences. Using the conservative Benjamini and Hochberg
(Benjamini and Hochberg, 1995) test and limiting the false discovery rate to 0.05 yielded a result of no significant differences between conditions. Due to the large number of probes that failed to hybridize because of sequence mismatches, another less stringent statistical approach was done (numerous published studies have used this approach as well). Genes were filtered for those showing a 50% change across conditions. Any genes detected with this method were tested for differences using a t-test. This approach yielded a list of nine genes that potentially increased in response to light and eight decreased.
Both c-fos and egr1 were found on the upregulated list. 18
Method 2a
Re-analysis of microarray data using MAS 5.0 method
There appeared to be an acceptably high level of false positives with the
first method of statistical analysis. The data was re-analyzed using the MAS 5.0
algorithm. In theory this method should be less sensitive to poorly hybridizing
probes withing a probe set.
Method 2b
Further refinement of MAS 5.0 microarray analysis
To further investigate the arrays, additional filters were added to restrict
analysis to those probe sets with a higher proportion of probes where the signal from the perfect match probe was significantly higher than the mismatch probe.
A filter was applied prior to the calculation of signal intensities. The signal
intensity from the perfect match probe was required to be at least 20 units higher
than the mismatch probe on at least 3 of the 12 genechips, otherwise, the probe
was excluded from all further calculations.
Results
We used laser capture microscopy (Figure 2-1) to isolate the SCN from
hamsters following treatment with either a light pulse or a sham light pulse. This
occurred 30 minutes after the start of treatment. After RNA from the tissue was
isolated and purified, the quality of the RNA was checked on an Agilent
Bioanalyzer so that had an 18s/28s RNA ratio close to 1.0 were processed
further (Figure 2-2). This ratio is indicative of good quality of sample. Samples 19
Figure 2-1 Laser Capture Microscopy Top: Hemotoxylin-stained section including the SCN. The line marks the area set for capture. Middle: Section after capture of the SCN. Appears darker than A because the picture in A was taken with xylene which provides greater clarity of image. Bottom: Images of three consecutive SCN sections on the capture device before RNA extraction. 20
Figure 2-2 Bioanalyzer verification of RNA quality The two dark bars in lanes 1-4 represent 18s and 28s rRNA. A ratio of 18s/28s rRNA close to 1.0 or better, along with the presence of two clear, undegraded bands of rRNA in the sample indicate good quality samples. 21
Figure 2-3 Hybridization Hybridization of hamster SCN RNA to an Affymetrix 430A 2.0 genechip. 22
were then hybridized to a Mouse 430A 2.0 Genechip (Figure 2-3), and we looked for differences in expression between the two treatments. Originally we found no significant differences but altering Method 1 for statistical analysis we were able to detect nine genes upregulated by light and eight detected as being downregulated (Table 2-1).
Due to problems finding primers to work for all of the genes identified, only
5 genes were able to yield results for qPCR validation (Table 2-2). The arrays
were able to correctly identify two genes previously known to be induced by light,
Egr1 and Fos. Egr1 was upregulated 30.17 fold after light pulse compared to
sham and Fos was upregulated 5.2 fold. Three genes that the microarrays
detected as being downregulated after light pulse, Cdh2, Hipk3, and Ppp2r5c
were found to have no significant difference between light and sham pulse with
fold changes of 1.33, 1.04 and 1.05 respectively. To make sure that the primers
were amplifying the correct sized amplicon, PCR products were run on the
Agilent Bioanalyzer to check for correct size (Figure 2-4). To see how similar the microarrays were, we made condition trees to see if the arrays would sort out according to treatment. We did this with Fos and Egr1 as seen in Figure 1-5.
The arrays were correctly sorted by treatments, showing that the microarrays were correctly identifying each treatment as two distinct groups.
Due to the number of high false positives we seemed to be getting, we re-
analyzed our data as described as Method 2a. Plots of the light pulse against
the dark pulse were made (Figure 2-6) and they show how before statistical 23
Table 2-1. Upregulated Genes Downregulated Genes Egr1 early growth response 1 Cdh2 cadherin 2 Fos FBJ osteosarcoma oncogene Za20b3 zinc finger, A20 domain containing 3 cytochrome c oxidase, subunit B-cell translocation gene 1, anti- Cox6b1 Btg1 VIb polypeptide 1 proliferative Homeodomain interacting protein Eno1 enolase 1, alpha non-neuron Hipk3 kinase 3 PRP19/PSO4 homolog (S. origin recognition complex, subunit 3- Prp19 Orc3l cerevisiae) like (S. cerevisiae) barrier to autointegration factor Protein phosphatase 2, regulatory Banf1 Ppp2r5c 1 subunit B (B56), gamma isoform hyaluronan mediated motility Hmmr Atg3l autophagy-related 3-like (yeast) receptor (RHAMM) Rho GDP dissociation inhibitor Arhgdia Doc2a double C2, alpha (GDI) alpha eukaryotic translation initiation Eif5a factor 5A
Genes found to be either upregulated or downregulated