UNIVERSITY OF CALIFORNIA SAN DIEGO

Homeodomain Transcription Factors VAX1 and SIX3 Modulate SCN Circadian Output

A Thesis submitted in partial satisfaction of the requirements for the degree Master of Science

in

Biology

by

Joseph A. Breuer

Committee in charge:

Professor Pamela L. Mellon, Chair Professor Brenda L. Bloodgood, Co-Chair Professor James W. Posakony

2018

Copyright

Joseph A. Breuer, 2018

All rights reserved. Signature Page

The Thesis of Joseph A. Breuer is approved and it is acceptable in quality and form for publication on microfilm and electronically:

Co-Chair

Chair

University of California San Diego

2018

iii Dedication

I would like to dedicate this thesis to my parents and grandparents for their constant love

and support, as well as to the many mice who sacrificed their lives for this project.

iv Table of Contents

Signature Page ...... iii Dedication ...... iv Table of Contents ...... v List of Figures and Tables ...... vii Abbreviations ...... viii Acknowledgements ...... x Abstract of the Thesis ...... xi Introduction ...... 1 Circadian rhythms are involved in our daily lives ...... 1 Suprachiasmatic nucleus and the mammalian molecular cock ...... 1 Homeodomain transcription factors, VAX1 and SIX3, are necessary for proper SCN development ...... 4 VAX1 and SIX3 are important for proper SCN function ...... 6 Vax1Syn mice, but not Six3Syn mice, have reduced VIP expression ...... 11 Study aims and goals ...... 11 Materials and Methods ...... 13 Cell Culture ...... 13 Transient Transfections ...... 13 Luciferase Assay ...... 14 Mouse Breeding ...... 14 Determination of Estrous Cyclicity ...... 15 Tissue Dissections ...... 16 LumiCycle ...... 16 Site-Directed Mutagenesis ...... 17 Results ...... 19 VAX1, but not SIX3, modulates Vip-luciferase expression via interaction with ATTA sites ...... 19 Six3Syn:Per2Luc mice have a longer Per2::luciferase period than Six3 wildtype Per2Luc mice ...... 21

v Vax1Syn:Per2Luc mice have a longer Per2::luciferase period than Vax1 wildtype Per2Luc mice ...... 23 VAX1 and SIX3 both modulate Per2-luciferase expression in vitro ...... 23 Mutating ATTA sites within Per2’s regulatory region alters the ability of VAX1, but not SIX3, to modulate Per2::luciferase expression ...... 26 Discussion ...... 31 VAX1, but not Six3, regulates Vip expression ...... 31 Vax1 and Six3 conditional knockout mice exhibit abnormal circadian period ...... 33 VAX1 modulates Per2 expression, while SIX3 does not ...... 34 SIX3 may directly regulate other molecular clock in the SCN ...... 35 Other potential pathways through which SIX3 or VAX1 may influence circadian rhythms ...... 35 Sex differences in circadian rhythms between male and female mice ...... 36 In conclusion ...... 37 References ...... 39

vi List of Figures and Tables

Figure 1. Suprachiasmatic Nucleus Input and Output ...... 2 Figure 2. The Mammalian Molecular Clock ...... 3 Figure 3. Vax1 Knockout Mice Lack Abnormal SCN Morphology ...... 5 Figure 4. Vax1 and Six3 Expression Localized to the Mouse SCN during Adulthood ...... 6 Figure 5. Vax1 Conditionally Knocked Out in Vax1flox/flox:SynapsinCre Mice ...... 7 Figure 6. Vax1Syn and Six3Syn have Abnormal Free-Running Activity ...... 8 Figure 7. Six3 Conditionally Knocked Out in Six3flox/flox:SynapsinCre Mice ...... 9 Figure 8. Vax1Syn, but not Six3Syn, Mice Reduced SCN VIP Expression ...... 11 Figure 9. VAX1 Increases Vip-luciferase Expression In Vitro ...... 20 Figure 10. VAX1’s Effect on Vip-luciferase Expression is Mitigated by Mutating ATTA Sites ...... 22 Figure 11. Six3Syn:Per2Luc Mice have a Longer SCN Per2 Period ...... 24 Figure 12. Vax1Syn:Per2Luc Mice have a Shorter SCN Per2 Period ...... 25 Figure 13. VAX1 and SIX3 Modulate Per2 Expression In Vitro ...... 27 Figure 14. Mutating ATTA Sites in the Per2 Promoter Alters VAX1-Mediated Expression ...... 29 Table 1. Mice Genotyping Primers ...... 15 Table 2. List of Primers used for Site-Directed Mutagenesis of Vip ...... 17 Table 2. List of Primers used for Site-Directed Mutagenesis of Vip ...... 18

vii Abbreviations

Avp Arginine vasopressin

Avpr1 Arginine vasopressin 1

Arntl/Bmal1 Aryl hydrocarbon receptor nuclear translocator-like

1

Cry1/2 Cryptochrome 1/2

E Embryonic day

EMSA Electromobility shift assay

GnRH Gonadotropin Releasing Hormone

H&E Hematoxylin and eosin

HPG Hypothalamic-Pituitary-Gonadal

IHC Immunohistochemistry

ISH In-situ hybridization

LacZ Beta galactosidase

Lhx1 LIM 1

Per1/2/3 Period 1/2/3

Per2Luc Per2::luciferase mice

Per2-Luc Per2-luciferase

SCN Suprachiasmatic nucleus

Six3 Six homeobox 3

Six3Syn Six3flox/flox:SynapsinCre mice

Six3Syn:Per2Luc Six3flox/flox:SynapsinCre:Per2Luc mice

viii Six6 Six homeobox 6 tPer2-luc Truncated Per2-luciferase

Vax1 Ventral anterior homeobox 1

Vax1Syn Vax1flox/flox:SynapsinCre mice

Vax1Syn:Per2Luc Vax1flox/flox:SynapsinCre:Per2Luc mice

Vip Vasoactive intestinal peptide

Vip-Luc Vip-luciferase

Per2-Luc Mutated Per2-luciferase

Vip-Luc Mutated Vip-luciferase

ix Acknowledgements

I would like to acknowledge Pamela L. Mellon, Ph.D., and Hanne M Hoffmann,

Ph.D. for endless amounts of guidance and encouragement throughout my time in the

Mellon Lab.

I would also like to thank my committee members, Brenda L. Bloodgood, Ph.D., and James W. Posakony, Ph.D. for their support and interest in my research.

All data on Six3 presented in this thesis are currently be being prepared for publication. I would like to thank the following co-authors: Jason D. Meadows, Hanne

M. Hoffmann, Erica L. Schoeller, Erica C. Pandolfi, Haley J. Oosterhouse, David R.

Natale, Michael R. Gorman, David K. Welsh, and Pamela L. Mellon. The thesis author is the third author on this paper.

Additionally, all data on Vax1 presented in this thesis are currently being prepared for publication. I would like to thank the following co-authors: Hanne M. Hoffmann,

Austin Chin, Jessica S. Lee, Crystal Trang, Jason D. Meadows, Brittany Hereford,

Michael R. Gorman, David K. Welsh, and Pamela L. Mellon. The thesis author is the second author on this paper.

x

ABSTRACT OF THE THESIS

Homedomain Transcription Factors VAX1 and SIX3 Modulate SCN Circadian Output

by

Joseph A. Breuer

Master of Science in Biology

University of California San Diego, 2018

Professor Pamela L. Mellon, Chair

Professor Brenda L. Bloodgood, Co-Chair

Circadian rhythms control many aspects of our daily lives: from waking and sleeping, to cell cycling within the body’s tissues. The suprachiasmatic nucleus (SCN) is often referred to as the body’s master pacemaker due to its ability to receive light information from the optic nerve and modulate a transcriptional-translational feedback loop, known as the molecular clock, and project circadian information from the SCN to the rest of the body via neuropeptide regulation. VAX1 and SIX3 are two homeodomain transcription factors, known to regulate expression, that are highly expressed in the

xi mature mouse SCN. Through the use of transgenic mouse models, we developed conditional Vax1 and Six3 knockout mice bred with Per2-luciferase reporters, a molecular clock gene, to monitor the effects of VAX1 and SIX3 deletion on SCN circadian output. We find that deletion of Vax1 or Six3 from the adult mouse SCN results in shorter and longer circadian period, respectively. Additionally, we determine the ability of VAX1 and SIX3 to increase the expression of Per2-luciferase in vitro, and additionally the ability of VAX1 to drive Vip-luciferase expression, the primary SCN neuropeptide responsible for projecting circadian information from the SCN to the body.

Homeodomain transcription factors are known to regulate gene expression by directly binding to ATTA sites within the gene’s promoter. We identified ATTA sites within regulatory regions of Per2 and Vip genes as potential targets for direct VAX1 interaction and regulation of gene expression. Overall, these data collectively identify VAX1 and

SIX3 as novel that are responsible for maintaining proper circadian rhythms during adulthood.

xii Introduction

Circadian rhythms are involved in our daily lives

The physical world is defined by a scale of rhythmic oscillations. Once a year,

Earth orbits 360° around the sun, and every 24 hours, Earth rotates 360° around its own axis. As mammals, many of our daily activities are also identified by such rhythmic profiles. From sleep and hunger, to hormone activity and cell cycle, many functions fluctuate in organic 24-hour cycles known as circadian rhythms (1-5). However, with so many aspects of life under the influence of circadian rhythms comes the caveat that several issues are capable of altering them. With problems such a shift work, working beyond the common 9 A.M. to 5 P.M., jetlag, and artificial lighting becoming much more common in everyday life, issues arise related to the maintenance of proper circadian rhythms (6-9). Reproductive health issues, cardiovascular morbidity, as well as gastrointestinal diseases have all been linked to having circadian origins, with progressively more links between common disorders and circadian rhythms being established every year (6,10). Thus, understanding the molecular basis for the generation and maintenance of circadian rhythms is becoming an ever-more important topic in biomedical research.

Suprachiasmatic nucleus and the mammalian molecular cock

The suprachiasmatic nucleus (SCN), a small region of the anterior hypothalamus, superior to the optic chiasm, is often referred to as the master circadian clock (11). Per its location adjacent to the optic chiasm, the SCN receives environmental input from the

1 eyes and functions within a light-dark cycle (Figure 1) (12). Light is sensed by optic receptors in the retino-ganglion cells, and is passed down the optic nerve to the SCN at the optic chiasm (13-15). Light signal triggers the upregulation of both aryl hydrocarbon nuclear translocator-like protein 1 (Arntl), also known as Bmal1, and Circadian locomotor output cycles kaput (Clock) (Figure 2) (16). BMAL1 and CLOCK dimerize and translocate to the nucleus where they bind to the enhancer box (E-box) within the promoters for the Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes and drive their expression (17-20). Lack of light reduces this process and triggers parasympathetic processes that result in pineal gland-secretion of melatonin and promote sleep (21). PERs and CRYs dimerize and translocate to the nucleus and bind the

BMAL1/CLOCK dimer and trigger its degradation (22,23). This negative feedback repression of PER/CRY is necessary for proper molecular clock function in the SCN

(24). Reduced BMAL1/CLOCK results in diminished expression of Per and Cry, and

Figure 1. Suprachiasmatic Nucleus Input and Output Light information is passed from the optic nerve to the suprachiasmatic nucleus (SCN) at the optic chiasm. VIP-expressing neurons have their cell bodies within the core SCN. VIP neurons project their axons to other hypothalamic neuronal populations as well as to AVP-expressing neurons in the shell of the SCN. AVP neurons relay light information from VIP neurons to surrounding hypothalamic neuronal populations as well as into the posterior pituitary.

2

Figure 2. The Mammalian Molecular Clock Aryl hydrocarbon nuclear translocator-like protein 1 (BMAL) and circadian locomotor output cycles kaput (CLOCK) dimerize and drive the expression of period (Per) and cryptochrome (Cry) via binding of the E-box. PER and CRY dimerize and translocate to the nucleus where they inhibit BMAL/CLOCK driven transcription. This results in a negative transcriptional-translational feedback loop capable of sustaining itself in approximately a 24-hour cycle.

3 thus, this negative feedback loop is able to sustain itself in a 24-hour cycle in the absence of light (25,26).

The SCN is composed of numerous different populations of neurons, including several GABAergic neurons populations with high neuropeptide expression (27).

Vasoactive intestinal peptide (VIP)-expressing neurons make up the largest portion of these neuropeptide-expressing cells within the suprachiasmatic nucleus, with their cell bodies positioned within the core of the SCN and their axons projecting outwards from the SCN (Figure 1). This specific orientation and positioning makes VIP-expressing neurons a key liaison for transmitting light and circadian information from the SCN to the rest of the body (28,29). In fact, full body knockout of Vip leads to abnormal free- running activity due to lack of proper SCN circadian output (30). VIP neurons also project their axons to arginine vasopressin (AVP) neurons, another neuropeptide- expressing cell population within the SCN that project their axons out of the SCN, also playing a key role in transmitting circadian information to the body (31,32).

Homeodomain transcription factors, VAX1 and SIX3, are necessary for proper SCN development

Homeodomain transcription factors are those that contain a characteristic homeodomain motif, allowing them to bind to DNA (12,33). It has been shown that homeodomain transcription factors preferably bind DNA at ATTA sites within the regulatory regions of genes, modulating their transcriptional activity (34,35). Ventral anterior homeobox 1 (Vax1) is a homeodomain that is required for development of the ventral forebrain and the eyes, as well as for contralateral motor and

4 cognitive activity (16,36,37). During mouse development, Vax1 is first expressed around embryonic day 8 (E8) with high localization near the anterior neural crest during neurulation (16). Vax1 knockout mice have several morphological defects, including holoprosencephaly and cleft lip, that contribute to perinatal death due to an inability to suckle (12). Additionally, our lab found that Vax1 knockout pups lack proper SCN morphology, suggesting VAX1 plays a key role in SCN development during embryogenesis (Figure 3, Hoffmann et al., in preparation). A downstream target of

VAX1 is Six homeobox 3 (SIX3). SIX3 is a second homeodomain transcription factor that is also important for proper embryonic development. In mice, SIX3 is first expressed around embryonic day 8.5 (E8.5) near the anterior neural plate, playing a significant role in both forebrain development, morphogenesis, and development of the optic nerve

(38-40). Similar to Vax1 knockout mice, Six3 knock out mice have significant morphological defects and do not survive birth (41).

A B

Figure 3. Vax1 Knockout Mice Lack Normal SCN Morphology H&E staining of the brain at birth (P0) in wildtype (A) and Vax1 knockout (B) mice. Arrow indicates location of the SCN. Wildtype mice have a denser cell population characterizing the SCN, while the cell density is absent in Vax1 knockout mice, indicating a lack of a proper SCN formation.

5 A B

SCN SCN

Figure 4. Vax1 and Six3 Expression Localized to the Mouse SCN during Adulthood In-situ hybridization on sagittal brain sections showing Vax1 (A) and Six3 (B) in the adult mouse brain (images from www.brain-map.org). Red arrow indicates location of Vax1 and Six3 in the SCN.

VAX1 and SIX3 are important for proper SCN function

During adulthood, Vax1 expression in the mouse brain is highly localized to the hypothalamus, with the densest expression in the SCN (Figure 4). To understand the role of Vax1 in mature neurons, our lab developed a Vax1 conditional knockout mouse line via crossing Vax1flox/flox mice with a SynapsinCre line to generate Vax1flox/flox:SynapsinCre

(Figure 5, Vax1Syn) mice. Synapsin is a phosphoprotein expressed in vesicles at synaptic terminals in mature neurons in both the central and peripheral nervous systems, allowing us to conditionally delete Vax1flox/flox only during post-natal neuronal maturation (42).

SynapsinCre in our Vax1Syn mice successfully targets deletion of Vax1 from the suprachiasmatic nucleus.

To determine if Vax1Syn mice had impaired SCN function, we evaluated wheel- running activity, a circadian behavior controlled by the SCN (Hoffmann et al., in preparation). In a 12-hour light:12-hour dark cycle, Vax1Syn mice had similar wheel-

6

Figure 5. Vax1 Conditionally Knocked Out in Vax1flox/flox:SynapsinCre Mice SynapsinCre mice were bred with Vax1flox/flox mice in order to generate Vax1flox/flox:SynapsinCre (Vax1Syn) mice. Synapsin is expressed in mature neurons and thus conditionally knocks out Vax1 from the SCN in late development.

7 B20 Cnts Scale: 0.2 220.0 B08 Cnts Scale: 0.2 279.5 B20 Cnts Scale: 0.2 220.0 0 8 16 0 8 16 0 0 8 16 0 8 16 003/02/15 0 8 16 0 8 16 0 B08 Cnts Scale: 0.2 279.5 03/02/15 07/26/14 Wildtype Vax1Syn Six3Syn 0 8 16 0 8 16 003/11/1503/11/15 08/04/1407/26/14 12-hr light: 08/04/14 03/21/1503/21/15

12-hr dark 08/14/14 03/31/1503/31/15 08/24/14 Constant 08/24/14 04/10/15 Darkness 09/03/14 04/10/15 09/03/1409/13/14 04/20/15 Figure 6. Vax1Syn and Six3Syn have Abnormal04/20/15 Free-Running Activity in Constant Darkness 09/13/1409/23/14 04/30/15 10/03/14Syn Syn 04/30/15 Wildtype, Vax1 , and Six3 mice housed05/10/15 in a 12-hour light:12-hour dark light cycle maintain a steady09/23/1410/13/14 wheel-running period of Tau = 24 hours since light re-entrains the molecular clock. Wildtype mice are able05/10/1505/20/15 to maintain a 23.5 hour period of free-running activity when 10/03/1410/23/14housed in constant darkness. Vax1Syn mice exhibited a shorter period, 05/30/15 while Six3Syn have11/02/14 a longer period, of 05/20/15free-running activity when housed in constant 10/13/14 darkness for 3 weeks. 06/09/15 05/30/15 10/23/14 running activity as wildtype mice, with06/09/15 an average period of 24 hours. This shows these 11/02/14 mice have normal light input to the SCN and are able to entrain to light. Placing mice in constant darkness allows us to reveal the endogenous free-running period (Tau) of the

SCN. In wildtype mice, the SCN free-running period is ~23.5 hours, leading to a slightly earlier activity onset every day when in constant darkness (Figure 6, constant darkness).

When placing Vax1Syn mice in constant darkness, a more sporadic, less periodic free- running activity was observed, with a general trend of shortening period (Figure 6). This shows VAX1 within the SCN is required for normal SCN output and behavioral circadian rhythms.

Parallel to Vax1, Six3 is also highly expressed in the adult mouse SCN (Figure

4B). Similarly, our lab generated a Six3 conditional knockout mouse line by breeding a

SynapsinCre mouse with Six3flox/flox mouse, producing our Six3flox/flox:SynapsinCre (Six3Syn) line (Figure 7A). To confirm successful deletion of Six3 within the SCN in situ

8 A

B Wildtype Six3Syn

Figure 7. Six3 Conditionally Knocked Out in Six3flox/flox:SynapsinCre Mice SynapsinCre mice were bred with Six3flox/flox mice in order to generate Six3flox/flox:SynapsinCre (Six3Syn) mice. Synapsin is expressed in mature neurons and thus conditionally knocks out Six3 from the SCN during late development. (B) In-situ hybridization for Six3 showing SynapsinCre successfully knocks out Six3 from the adult mouse SCN.

9 hybridization for Six3 was done. Six3Syn had >90% reduction in Six3 transcript levels in the SCN (Figure 7B). To determine if Six3 in the adult SCN was important for SCN output, control and Six3Syn mice were placed in running wheels. Six3Syn mice displayed normal wheel-running activity when housed in a 12-hour light:12-hour dark cycle with an average free running period of 24 hours. However, like Vax1Syn mice, Six3Syn mice exhibit a more sporadic free-running activity when housed in constant darkness, with an initial shorter free running period, which became longer after prolonged time in constant darkness (3 weeks, Figure 6). In contrast, all controls had a free running period <24-hr in constant darkness.

The abnormal free-running activity of both our Vax1Syn and Six3Syn mice when housed in constant darkness is the result of one of two possibilities. It is possible that

VAX1 or SIX3 are necessary for proper SCN molecular clock gene expression: Per, Cry,

Clock, and/or Bmal. Thus, absence of VAX1 or SIX3 may alter the periodicity of the negative feedback-inhibition loop that maintains circadian rhythms in the absence of light

(Figure 2). The other possibility is that VAX1 or SIX3 may alter SCN neuropeptide expression. SCN neuronal populations, including VIP+ and AVP+ neurons, are largely responsible for transmitting circadian information from the SCN to the rest of the body

(Figure 1). If Vip or Avp expression is normally modulated by VAX1 or SIX3, absence or alteration of either neuropeptide output from the SCN may have drastic effects on SCN circadian control of periodic functions, including sleep vs. waking cycles.

Vax1Syn mice, but not Six3Syn mice, have reduced VIP expression

To determine if VAX1 or SIX3 regulate Vip expression in the SCN,

10 immunohistochemistry labelling for VIP was performed. Interestingly, Vax1Syn mice have reduced VIP expression in the SCN compared to wildtype mice, indicating that

VAX1 is required for normal VIP expression (Figure 8). However, Six3Syn mice exhibited a normal amount of VIP expression compared to wildtype mice, suggesting that

SIX3 is not necessary for VIP expression. Overall, this indicates that VAX1 is likely regulating circadian output from the SCN by modulating VIP expression.

Study aims and goals

Our goal is to determine how VAX1 and SIX3 are regulating circadian rhythms in the SCN. Vax1Syn and Six3Syn conditional knockout mice both exhibited abnormal free- running activity when housed in constant darkness, indicating that lack of VAX1 or SIX3 from the adult SCN leads to abnormal circadian rhythms. This is likely due to VAX1 and

SIX3 either directly regulating the expression of molecular clock genes or through

A B Wildtype Vax1Syn Wildtype Six3Syn

Figure 8. Vax1Syn, but not Six3Syn, Mice have Reduced SCN VIP Expression Immunohistochemistry labelling of the SCN for VIP in both Vax1Syn (A) and Six3Syn (B) ice. Vax1Syn mice have significantly less VIP expression relative to wildtype mice, while Six3Syn mice have normal amounts of VIP expression, suggesting that VAX1, but not SIX3, regulate Vip expression.

11 regulation of SCN neuropeptide expression, which would affect the SCN’s ability to project circadian information to the rest of the body. In fact, Vax1Syn mice have reduced

VIP expression in the SCN, suggesting that VAX1 may regulate circadian output through regulation of VIP expression. However, VAX1 may also be regulating molecular clock gene expression. Additionally, Six3Syn mice exhibited wildtype levels of VIP expression, suggesting SIX3 may regulate circadian output via modulating alternate SCN neuropeptide expression, or through direct regulation of molecular clock gene expression.

Therefore, our first goal is to confirm that VAX1 is capable of regulating Vip expression in vitro and to see if we can identify if VAX1 is interacting with Vip’s regulatory region.

Second, we want to explore whether VAX1 and SIX3 are altering molecular clock gene expression and if they are doing so via direct interaction with the promoter regions of the molecular clock genes.

My hypothesis is that VAX1 and SIX3 regulate SCN circadian output via direct

modulation of molecular clock gene or SCN neuropeptide expression.

12 Materials and Methods

Cell Culture

NIH-3T3 mice fibroblasts (American Type Culture Collection) were cultured in

Dulbecco’s Modified Eagle Medium (DMEM) (Corning Cellgro) with 10% fetal bovine serum (FBS) (Omega Scientific) and 1% penicillin-streptomycin at 37°C and humidified

CO2. Cells were split every 2-3 days once 90% confluency was achieved.

Transient Transfections

NIH-3T3 cells were plated at 6,000 cells per well in 24-well plates for luciferase assay using PolyJet (SigmaGen Laboratories) in DMEM. Cells were co-transfected with

150 ng/well Vip-full length promoter in pGL3-basic backbone from Hatori et al., 2014

(43), referred to as Vip-Luc, truncated Per2 promoter (Per2: -1128 to -141), referred to as tPer2-Luc, or full-length Per2 promoter containing exon 1 and intron 1 (Per2: -1128 to

+2129), referred to as Per2-Luc from Yoo et al., 2005 (20). 100 ng/well thymidine kinase--galactosidase reporter plasmid used as a control for transfection efficiency.

Cells were co-transfected with expression vectors containing 20 ng/well of Vax1 in a pCMV6 backbone (CMV, True Clone, Origene) or 200 ng/well of Six3 in a pSG5 backbone from Larder et al., 2013 (44). Cells were transfected approximately 24-hrs after plating in 24-well plates at a concentration of 6,000 cells per well. Mean confluency was

55% at time of transfections. Media was removed 8-hrs post transfection and replenished with fresh DMEM with 10% FBS and 1% penicillin-streptomycin.

13 Luciferase Assay

Transfected cells were harvested approximately 24-hrs post transfection in lysis buffer (100 mM potassium phosphate, pH 7.8, and 0.2% Triton X-100). Luciferase and

-galactosidase assays were performed. Luciferase assay results were normalized to their respective -galactosidase assay values for standardization for transfection efficiency.

Luciferase/-galactosidase values were then normalized to pGL3-basic luciferase backbone activity to control for baseline backbone expression.

Mouse Breeding

All animal procedures were performed in accordance with the UCSD Institutional

Animal Care and Use Committee (IACUC) regulations. Mice used in this thesis were group-housed on a 12-hr:12-hr light/dark cycle and given ad libitum chow and water.

Vax1flox/flox mice generated in Hoffmann et al., 2016, were used to generate Vax1 conditional knockout mice. To generate mice with a conditional knockout of Vax1,

Vax1flox/flox mice were crossed with SynapsinCre mice from Zhu et al., 2001, (45) to generate Vax1flox/flox:SynapsinCre (Vax1Syn) mice. Vax1Syn mice were crossed with

Per2::luciferase mice (Per2Luc) to generate triple-transgenic

Vax1flox/flox:SynapsinCre:Per2Luc (Vax1Syn:Per2Luc) mice. Six3flox/flox mice from Oliver et al., 1995, were used in order to generate Six3 conditional knockout mice. To generate mice with a conditional knockout of Six3, Six3flox/flox mice were cross with SynapsinCre mice to generate Six3flox/flox:SynapsinCre (Six3Syn) mice. Six3Syn mice were crossed with

Per2Luc to generate triple-transgenic Six3flox/flox:SynapsinCre:Per2Luc (Six3Syn:Per2Luc) mice. All mice were kept on a C57BL background. Mice were sacrificed by either CO2

14 or isoflurane inhalation with cervical dislocation. Mice were genotyped using primers presented in Table 1.

Table 1. Mice Genotyping Primers Table of the primers used to genotype all mice used in this thesis using PCR and agarose gel electrophoresis. SynapsinCre has a tendency to recombine floxed genes in the germline, thus primers were designed to determine recombination.

Gene Primer Primer Sequence (5`→ 3`) Anneal T Name (°C) SynapsinCre CreOlef F GCATTACCGGTCGTAGCAACGAGTG 60 CreOlef R GAACGCTAGAGCCTGTTTTGCACGTTC Vax1 Wt F CCAGTAAGAGCCCCTTTGGG Vax1flox Vax1 Flox F GCCGGAACCGAAGTTCCTA 55 Vax1 R CGGATAGACCCCTTGGCATC Six3 Wt F TTCCCCTCTTTGACTCCTATGGACG Six3flox Six3 Flox F CGGCCCATGTACAACGCGTATT 59 Six3 R CCCCTAGCCTAACCCAAACATTCG Per2::luciferase Per2 F CTGTGTTTACTGCGAGAGT Per2 Wt R GGGTCCATGTGATTAGAAAC 60 Per2 Mut R TAAAACCGGGAGGTAGATGAGA Vax1flox Germ- VF2 F GCAGTGGCCTAGAGAGATCG Line VF2 R CCTGGCGCCCTACAAACATA 55 Recombination VF Wt R CAGACACCGGAGGAGGAAAC

Determination of Estrous Cyclicity

To establish estrous cyclicity for determination of mice fate, vaginal smears were performed on 12-20 week-old mice by vaginal lavage with 50 μL sterile MQ water.

Smears were collected on glass slides and observed through bright-field microscopy

(determination before death) or counterstained with 0.1% methylene blue (Spectrum,

Gardena, CA) before microscopy (determination after death) to determine corresponding stage of estrous cyclicity based on cellular phenotype.

15 Tissue Dissections

Mice were sacrificed in the morning via isoflurane inhalation and cervical dislocation (mean time: 10:15 A.M.). Blood and tail clippings were collected for hormone level determination and genotyping, respectively. Tissues were immediately removed from the mice and placed in an ice slushy HBSS solution on ice for approximately 1 hr. Brains were removed, maintaining the optic chiasm. Slices of the brain were obtained using the Vibratome. Brains were trimmed into a 5-6 mm block containing the hypothalamus on both sides. The trimmed brains were loaded onto the specimen holder with the anterior side facing up and the optic chiasm resting against a cube of 2% agarose gel. The holder and hypothalamic tissue were placed in the slicing tray filled with 80 mL of half frozen HBSS and surrounded by ice (refilled as ice melted).

150 μm thick coronal brain slices were obtained using a Leica Vibratome. Brain slices were stored in Neurobasal-A Medium (Gibco by Life Technologies) with L-Glutamine

(Glutamax) and B-27 supplement (source). The suprachiasmatic nuclei (SCN) were dissected in 2x2 mm squares from 0.3 mm coronal slices corresponding to -0.30 to -0.80 bregma.

LumiCycle

35 mm tissue plates contained 1 mL of Neurobasal-A Medium (Gibco) with 1%

Glutamax (Gibco), 1% B-27 supplement (Gibco), and 1% sterile Firefly D-luciferin (BD

Biosciences). Suprachiasmatic nuclei were dissected (mean time: 11:30 A.M.) and placed on Millicell membranes (Millepore: Millicell Cell Culture Inserts) and placed inside the 35 mm tissue plates. Lids were sealed to the plates using vacuum grease to

16 ensure an air-tight seal. All plated tissues were then stored in a dark 35 °C incubator until all tissues were ready to be loaded into the LumiCycle. Plated tissues were loaded into the Lumicycle inside the 35 °C incubator simultaneously (mean time: 12:15 P.M.) and recordings were started. Recordings were taken every 0.07 days and preceded for approximately 1 wk.

Site Directed Mutagenesis

ATTA sites within Vip and Per2’s regulatory regions were identified using

Sanger Sequencing (Eton Bioscience Inc.) and using ApE-A Plasmid Editor to identify

ATTA sites. ATTA sites were confirmed in mouse DNA via NCBI BLAST. Site- directed mutagenesis primers were created using NEB Base Changer (Table 2 and 3).

NEB Q5 Site-Directed Mutagenesis Protocol

Table 2. List of Primers used for Site-Directed Mutagenesis of Vip Table of primers used to induce mutations at ATTA sites within the Vip promoter (Vip- pGL3) plasmid. Position refers to the number of base pairs upstream of the transcription start site. All primers were designed using NEBase Changer (https://nebasechanger.neb.com).

Position Mutation Primer Sequence Anneal T (°C) -909 ATTA → Forward TTAAGTCCCAcggcAATGTATGTCAGGCTAC 58 CGGC Reverse TGTCAAAATGCAGCTATC -862 ATTA → Forward ACAAAAAAAAcggcAGCATTCATCACAGTACTC 56 CGGC Reverse TGATTTTAACCTCAAAGTAGC -408 TAAT → Forward TAGAAAACTTgccgTCAGATATGCATAAGCAG 56 GCCG Reverse GAAACCTACTCTAATTTAAAAAG -365 ATTA → Forward TTTAAAAGGCcggcAAGCAATTGACACTTTG 56 CGGC Reverse AAACTTTATGCTGCTTATG -204 ATTA → Forward CTAAGTAAAGcggcAAGCATTGCACATC 56 CGGC Reverse CTGTGATGAACTAGAAGATG -94 TAAT → Forward TGATGACATTgccgAAGAACTTCAAGACC 58 GCCG Reverse TGCTGATTTATATACATTTATAGC

17 (https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale0554.pdf) was followed to induce mutations into ATTA. To confirm correct mutations sequences were sequenced using Sanger sequencing from Eton Biosciences. Tables of site directed mutagenesis primers are in Tables 2 and 3.

Table 3. List of Primers used for Site-Directed Mutagenesis of Per2 Table of primers used to induce mutations at ATTA sites within the Per2 promoter (pGL3) plasmid. Position refers to the number of base pairs upstream or downstream of the transcription start site. All primers were designed using NEBase Changer (https://nebasechanger.neb.com).

Position Mutation Primer Sequence Anneal T (°C) -717 TAAT → Forward CCTGTAAGGTgccgAAACTACACCACCG 63 GCCG Reverse AACTATGGAAGCGGGGTG -602 ATTA → Forward CTGCACGGCAcggcTGACCTTATTTCCTG 57 CGGC Reverse AGATACAAAGGAAAGAAGTG -316 ATTA → Forward GGTCCTTCGGcggcCCGAGCTGGTC 63 CGGC Reverse GAGAATGCTCTCTGCTGAC +366 ATTA → Forward GGTCGGAGAGgccgGTTAGGCATCTTG 60 GCCG Reverse TCAGTGCCTAACCTGAGA +1319 TAAT → Forward TCCTTTATTTcggcGGTAGCTGTACAGTG 59 CGGC Reverse GTCCCTGGCTTTCTTAGATTC +1774 TAAT → Forward GAGCCTATTAcggcGGTATAATTTCCTCCAAT CGGC CTCAG 60 Reverse CCATACCTCTCAGGGTTTTC

18 Results

VAX1, but not SIX3, modulates Vip-luciferase expression via interaction with ATTA sites

Since Vax1Syn mice have decreased VIP+ neurons in the SCN, we determined whether VAX1 could directly alter Vasoactive intestinal peptide (Vip) expression in vitro.

Therefore, NIH-3T3 mice fibroblasts, which maintain circadian clock rhythmicity in vitro, were co-transfected with a full-length Vip promoter/enhancer luciferase construct as well as either a Vax1-overexpression plasmid or a Six3-overexpression plasmid and assayed for Vip-luciferase expression 48 hours post transfection (Figure 9A-B). As expected, VAX1-overexpression successfully modulated Vip-luciferase expression, resulting in a nearly a three-fold increase in expression compared with co-transfection with empty vector (Figure 9C). Additionally, SIX3-overexpression did not significantly alter Vip-luciferase expression relative to co-transfection with empty vector alone, although a minor decreasing trend was observed, supporting the normal expression of

VIP seen in Six3Syn mice (Figure 8).

Homeodomain transcription factors, a family that includes VAX1, are known for directly interacting with gene-regulatory regions via ATTA sites. VAX1 has previously been shown to alter GnRH expression via binding of ATTA sites within the Gnrh promoter (35). Therefore, we wanted to determine if mutating ATTA sites within Vip’s promoter would mitigate VAX1-modulated increase in Vip-luciferase expression. To test this, we sequenced our Vip-luciferase construct and identified six ATTA sites within the full promoter (Figure 10A). Site-directed mutagenesis was utilized to develop Vip- luciferase plasmids for each individual mutation (primers in Table 2). Each mutation

19 A

B

C D

Figure 9. VAX1 Increases Vip-luciferase Expression In Vitro (A) Experimental design: NIH-3T3 mice fibroblasts co-transfected with Vip-luciferase and either Vax1 or Six3 overexpression plasmids. Luciferase activity recorded 48-hours post transfection. (B) Full-length Vip promoter/enhancer luciferase construct used for luciferase assays. Vip-luciferase expression results when co-transfected with either Vax1-overexpressing (C, n = 7) or Six3-overexpressing (D, n = 7) plasmids. Statistical analysis by Student’s t-test. **, p<0.01.

20 was then screened in a luciferase assay to observe which mutations could mitigate

VAX1’s effect on Vip-luciferase expression. The screening demonstrated that mutating the ATTA site at position -862 relative to the transcription start site showed potential as a site for this effect (Figure 10B). Follow up luciferase assay results demonstrated that co- transfection of Vip -862-luciferase with Vax1-overexpressing plasmid resulted in nearly half the expression than Vax1-overespressing plasmid co-transfected with non-mutated

Vip-luciferase alone (Figure 10C). This suggests that the ATTA site at position -862 is likely involved in VAX1-regulation of Vip expression, but not necessary.

Six3Syn:Per2Luc mice have a longer Per2::luciferase period than Six3 wildtype Per2Luc mice

Since SIX3 was not shown to modulate Vip expression either in vitro or in vivo, we wanted to determine if abnormal free-running activity seen in Six3Syn mice is due to

SIX3 modulation of SCN molecular clock gene expression. We cross-bred our Six3Syn mice with Per2::luciferase (Per2Luc) mice in order to generate

Six3flox/flox:SynapsinCre:Per2Luc (Six3Syn:Per2Luc) mice (Figure 11A).

To record Per2::luciferase expression in live tissue explants over time, suprachiasmatic nuclei were dissected from 12-20 week old Six3Syn:Per2Luc and wildtype

Six3 Per2Luc mice and cultured in dishes containing luciferin. SCN explants were placed in a LumiCycle to monitor luciferase activity (Figure 11B). Wildtype Per2Luc mice had an average SCN Per2::luciferase period of approximately 26.5 hours, while

Six3Syn:Per2Luc mice had an average period of around 28 hours (Figure 11C). This

21 A

B

C

Figure 10. VAX1’s Effect on Vip-luciferase Expression is Mitigated by Mutating ATTA Sites (A) Six ATTA sites were identified and mutated using site-directed mutagenesis within Vip’s promoter/enhancer region. (B) ATTA at position -365 relative to the transcription start site appeared to be significant in initial screening. (C) Luciferase assay results showing mitigation of VAX1-overexpression effect when co-transfected with Vip µ-862- luciferase. Number above Vax1-treated samples represents fold-change relative to respective mutants treated with empty vector. Statistical analysis using two-way ANOVA followed by Bonferroni Post-Hoc, comparing Vax1-treated mutants with Vax1- treated wildtype, showed no significance. N = 2-7.

22 suggests that Six3 expression in the adult mouse SCN is required for maintaining proper period of molecular clock gene expression.

Vax1Syn:Per2Luc mice have a longer Per2::luciferase period than Vax1 wildtype Per2Luc mice

We next crossed the Vax1Syn mice with the Per2Luc mice to generate

Vax1flox/flox:SynapsinCre:Per2Luc (Vax1Sym:Per2Luc) mice to observe the effect of Vax1 conditional knockout on SCN molecular clock gene expression (Figure 12A).

Suprachiasmatic nuclei were explanted from 12-20-week-old male and female

Vax1Syn:Per2Luc and Vax1 wildtype Per2Luc mice and cultured in dishes containing luciferin. Per2::luciferase expression was then monitored in the LumiCycle for seven days. Vax1SynPer2Luc showed circadian-like Per2::luciferase expression but with an average Per2 period of 25-hours, significantly shorter than Vax1 wildtype Per2Luc mice by approximately 1.5 hours (Figures 12B-C). This suggests that expression of Vax1 in the adult mouse SCN is necessary for maintaining proper molecular clock gene expression and circadian rhythms.

VAX1 and SIX3 both modulate Per2-luciferase expression in vitro

Now that we had established that conditional knockout of either Vax1 or Six3 from the adult mouse SCN is capable of altering rhythmicity of molecular clock gene expression, we wanted to determine if VAX1 and SIX3 are capable of altering Per2 expression in vitro. To do so, we analyzed Per2-luciferase expression in NIH-3T3 mouse fibroblast cells due to their ability to maintain circadian clock rhythmicity in tissue

23 A

B C

Figure 11. Six3Syn:Per2Luc Mice have a Longer SCN Per2 Period (A) Diagram of mating between Six3flox/flox:SynapsinCre mice and Per2Luc mice to generate Six3flox/flox:SynapsinCre:Per2Luc (Six3Syn:Per2Luc) mice. (B) Per2::luciferase expression over time in Six3Syn:Per2Luc and Per2Luc (wildtype) mice SCN explants. (C) Average Per2::luciferase period in Six3Syn:Per2Luc and Per2Luc (wildtype) mice SCN explants. Statistical analysis performed using Student’s t-test. **, p<0.01. N = 4-6.

24 A

B C

Figure 12. Vax1Syn:Per2Luc Mice have a Shorter SCN Per2 Period (A) Diagram of mating between Vax1flox/flox:SynapsinCre mice and Per2Luc mice to generate Vax1flox/flox:SynapsinCre:Per2Luc (Vax1Syn:Per2Luc) mice. (B) Per2::luciferase expression over time in Vax1Syn:Per2Luc and Per2Luc (wildtype) mice SCN explants. (C) Average Per2::luciferase period in Vax1Syn:Per2Luc and Per2Luc (wildtype) mice SCN. Statistical analysis performed using Student’s t-test. **, p<0.01. N = 4-6.

25 culture. NIH-3T3 cells were transfected with either a full-length Per2 luciferase reporter construct (Per2-Luc) or a truncated Per2 promoter (tPer2-Luc) (Figure 13A). To induce either VAX1 or SIX3 overexpression, the cells were additionally transfected with either

Vax1 or Six3-overexpression plasmids. Per2-luciferase activity was recorded 48-hours post transfection. VAX1 overexpression induced nearly a two-fold increase in Per2-Luc expression relative to treatment with empty vector. Surprisingly, not only was this

VAX1-induced increase in expression mitigated in the truncated Per2 promoter (tPer2-

Luc) but, actually, VAX1 overexpression resulted in approximately a two-fold decrease in luciferase activity (Figure 13B). Not surprisingly, Six3 overexpression resulted in similar patterns of regulation of Per2-Luc expression, although more dramatic, with SIX3 driving a fifteen-fold increase in Per2-luc expression, whereas SIX3 acted as a repressor for tPer2-Luc, decreasing tPer2-Luc expression by fifteen-fold (Figure 13B). In both cases, the data suggest that VAX1 and SIX3 are both capable of altering Per2-luciferase expression in vitro. Additionally, Per2 appears to have essential regulatory sequences present within intron 1 that may have an enhancing effect on Per2 expression in the presence of either VAX1 or SIX3.

Mutating ATTA sites within Per2’s regulatory region alters the ability of VAX1, but not

SIX3, to modulate Per2-luciferase expression

To test how VAX1 modulates Per2-luciferase expression in vitro, ATTA sites were identified within our Per2-Luc reporter constructed (Figure 14A) at several positions, all of which lie within highly conserved regions of Per2 (UCSC Genome

Browser). Site-directed mutagenesis was used to induce individual mutations at all eight

26

A

B C

Figure 13. VAX1 and SIX3 Modulate Per2 Expression In Vitro (A) Per2-luciferase reporter constructs used for luciferase assay. Luciferase assay results for the effects of VAX1 (B, n = 4-7) or SIX3 (C, n = 5) overexpression on both Per2-luc and tPer2 expression. Statistical testing was performed using Student’s t-test. **, p<0.01.

27 ATTA sequences located in the conserved regulatory sequences of the promoter and intron 1. Each mutation was then screened via co-transfection of NIH-3T3 cells with either Vax1 or Six3-overexpressing plasmids and either wildtype Per2-Luc or mutated

Per2-Luc. Forty-eight hours post transfection, Per2-luc expression was assayed, and it was determined that mutating the ATTA site at position +1770 relative to the transcription start site was capable of reducing VAX1-induced Per2-luciferase nearly two fold, while, surprisingly, mutation of the ATTA site at position +1774 relative resulted in complete mitigation of VAX1-induced increase in Per2-luciferase expression (Figure

14B). In contrast, none of the eight mutations appeared to alter the ability of SIX3 to increase Per2-luciferase expression, suggesting that Six3 may not alter Per2 expression via direct interaction (Figure 14C). Repeated NIH-3T3 co-transfections with Vax1- overexpression plasmid and Per2-Luc µ+1774 demonstrated that mutation of ATTA sequences at this position completely mitigated the VAX1-induced Per2-luciferase expression (Figures 14B-C), suggesting that this site is necessary for VAX1-regulation of

Per2 expression. Additionally, mutating ATTA at the adjacent position +1770 relative to the TSS resulted in decreased VAX1-induced Per2-luciferase expression, albite not significant. Overall, these data suggest that VAX1 is likely directly interacting with Per2 to modulate its expression.

28 A

B

C D

Figure 14. Mutating ATTA Sites in the Per2 Promoter Alters VAX1-Mediated Expression (A) Eight ATTA sites were identified and mutated using site-directed mutagenesis within Per2’s regulatory region (Per2-Luc). (B) Mutations generated at position +1774 and relative to the TSS. Luciferase assay results showing VAX1 (C, n = 2-6) and Six3 (D, n = 2-6) transcriptional effects on the indicated constructs. Statistical analysis performed using two-way ANOVA followed by Bonferroni Post-Hoc comparing Vax1-treated mutants with Vax1-treated wildtype. **, p<0.01.

29 All data on Six3 presented in this thesis are currently be being prepared for publication. I would like to thank the following co-authors: Jason D. Meadows, Hanne

M. Hoffmann, Erica L. Schoeller, Erica C. Pandolfi, Haley J. Oosterhouse, David R.

Natale, Michael R. Gorman, David K. Welsh, and Pamela L. Mellon. The thesis author is the third author on this paper.

Additionally, all data on Vax1 presented in this thesis are currently being prepared for publication. I would like to thank the following co-authors: Hanne M. Hoffmann,

Austin Chin, Jessica S. Lee, Crystal Trang, Jason D. Meadows, Brittany Hereford,

Michael R. Gorman, David K. Welsh, and Pamela L. Mellon. The thesis author is the second author on this paper.

30 Discussion

To provide further insight into the regulation of circadian rhythms and output of the SCN, we investigated the roles of the homeodomain transcription factors Vax1 and

Six3, which are highly expressed in the mouse SCN during adulthood, on circadian output. Due to abnormal wheel-running activity seen in both our Vax1flox/flox:SynapsinCre

(Vax1Syn) and Six3flox/flox:SynapsinCre (Six3Syn) mice, we hypothesized that VAX1 and

SIX3 directly modulate molecular clock gene or SCN neuropeptide expression in the

SCN to modulate SCN circadian output.

VAX1, but not Six3, regulates Vip expression

VIP neuropeptide expressing cells make up the greatest portion of cell bodies within the SCN and project their axons towards other brain regions, and thus are primary transmitters of SCN circadian information. Conditional deletion of Vax1 from the mature

SCN in Vax1Syn mice resulted in a significant reduction of vasoactive intestinal peptide

(VIP) in the SCN and a shorter free-running period in constant darkness compared to wildtype mice. Interestingly, this is comparable to the shortened free-running period seen in Vip knockout mice, further suggesting that Vax1 may be essential for regulating VIP expression in the SCN (46).

When observing the effect of VAX1 on Vip-luciferase expression in vitro, we found that VAX1 overexpression increases Vip expression likely through interacting with

ATTA sites within the promoter region. Interestingly, we found that VAX1-induced Vip expression was only partially mitigated via mutation of single ATTA sites within Vip’s

31 promoter. However, it is likely that if VAX1 directly binds to the Vip promoter, it interacts with several ATTA sites, so perhaps inducing mutations at multiple ATTA sites would allow for complete mitigation of Vip expression during VAX1 overexpression. It is also possible that VAX1 regulates Vip expression as a secondary transcription factor.

Therefore, to confirm whether VAX1 directly interacts with Vip at ATTA sites, we will perform an electro-mobility shift assay (EMSA) using VAX1 lysates and Vip ATTA oligos, allowing us to confirm where direct binding is specifically occurring.

Nevertheless, these results suggest that VAX1 is capable of modulating VIP expression, but whether or not this is via direct interaction with Vip or as a secondary transcription factor through modulation of other factors is yet to be determined.

Six3 is a second homeodomain transcription factor that is highly expressed in the mouse SCN during adulthood. Previous work in the Mellon lab had shown that conditional knockout of Six3 from the adult SCN in Six3Syn mice does not impact SCN

VIP expression. In agreement with this, we did not see any effect of SIX3- overexpression to regulate Vip-luciferase expression in vitro. However, SIX3 may alter circadian output via other SCN neuropeptide-expressing cells. Arginine vasopressin- expressing (AVP+) neurons are also highly abundant in the SCN, receiving input from

VIP+ neurons and projecting circadian information to the rest of the body. It was previously demonstrated that Six3Syn mice had a longer free-running period in constant darkness than seen in wildtype mice. Although no studies have observed how Avp knockout in mice alters free-running activity, Avpr1-/- (AVP receptor) knockout mice have a longer free-running activity in constant darkness (47). Additionally, Bmal1 conditional knockout from AVP-expressing neurons results in a longer SCN period (48)

32 Even though Six3Syn mice have wildtype levels of AVP expression, in correlation with increased free-running period seen in Six3Syn mice, it may be possible that knockout out

Avp would result in a similar phenotype as when inhibiting the molecular clock within

AVP+ neurons. Nevertheless, future experiments would be performed, observing the free-running activity phenotype of Avp knockout mice, as well as determining whether

Six3Syn or Vax1Syn mice have altered AVP expression in the SCN relative to wildtype mice.

Vax1 and Six3 conditional knockout mice exhibit abnormal circadian clock period

The molecular clock is composed of four primary genes that function together in a transcription-translation feedback loop: Clock, Bmal, Cry, and Per (Figure 2). Due to the negative-feedback inhibitory nature of this cyclical pathway, modulation of a single gene should result in a phenotype for all four genes. Vax1flox/flox:SynapsinCre:Per2Luc

(Vax1Syn:Per2Luc) mice SCN explants demonstrated a shorter PER2::luciferase period relative to Vax1 wildtype Per2Luc mice, while explants from

Six3flox/flox:SynapsinCre:Per2Luc (Six3Syn:Per2Luc) exhibited a longer SCN PER2::luciferase period. For Vax1Syn:Per2Luc mice, the shortened PER2::luciferase period coincided with the shortened period seen in the free-running activity in constant darkness of Vax1Syn mice. This confirms that, in addition to VAX1 modulating the expression profile of SCN neuropeptide gene Vip, VAX1 is also necessary for maintaining proper expression of the molecular clock genes within the SCN.

Similarly, the longer PER2::luciferase period seen in Six3Syn:Per2Luc mice SCN explants resembles the longer behavioral free-running period of Six3Syn mice in constant

33 darkness. This suggests that SIX3 modulates the SCN molecular clock cycle. In either case, however, this does not determine whether VAX1 or SIX3 are directly altering the expression of Per2, or another clock gene. Regardless, these data suggest that VAX1 and

SIX3 are necessary for the maintenance of normal circadian rhythms within the SCN.

VAX1 modulates Per2 expression, while SIX3 does not

Vax1 and Six3 expression are high in the adult mouse SCN. Conditional knockout of Vax1 and Six3 from the SCN in Vax1Syn and Six3Syn mice, respectively, results in abnormal free running activity in constant darkness, which is likely due to either modulation of SCN neuropeptide expression and circadian output, or direct modulation of molecular clock gene expression. Although we had already demonstrated that VAX1 is capable of modulating the expression of the SCN neuropeptide VIP, which is a possible explanation for the shortened free-running period in Vax1Syn mice, SIX3 does not alter

VIP expression, and thus, we wanted to determine if VAX1 and SIX3 can regulate the transcription of the molecular clock gene Per2. It was determined that both Vax1 and

Six3 overexpression increase Per2-luciferase expression in NIH-3T3 mouse fibroblasts.

These results correlate with the change in period of PER2::luciferase expression seen in

Vax1Syn:Per2Luc and Six3Syn:Per2Luc mice. However, it was surprising to see that VAX1 and SIX3 overexpression both lead to increased Per2-luciferase expression in vitro, since the results of the conditional knockout of Vax1 and Six3 on the SCN explant

PER2::luciferase period were opposite. Additionally, only VAX1-induced modulation of mouse Per2-luciferase is mitigated through mutating ATTA sites within Per2’s regulatory region, specifically at positions +1774 within intron 1. This suggests that

34 VAX1 is altering Per2 expression through this ATTA site. To confirm the direct binding of VAX1 to this site, we will perform EMSA’s in the future. Surprisingly, our results suggest that SIX3 is not directly altering Per2 expression. A possibility is that SIX3 modulates other transcription factors, which, in turn regulate Per2 or SIX3 might impact circadian rhythm generation through modulation of other clock genes.

SIX3 may directly regulate other molecular clock genes in the SCN

NIH-3T3 cells are often used in circadian studies because of their well-maintained circadian rhythmicity. The increase in Per2-luciferase expression seen during Six3- overexpression in NIH-3T3 cells may not be the result of SIX3 directly binding Per2, but rather through binding of another endogenous clock gene. Thus, in vitro studies exploring the effects of Six3-overexpression on other molecular clock-driving-luciferase reporters would be of interest. Additionally, three new sites resembling ATTA sites within intron 1 of Per2 have since been identified at positions +1183 and +1782. We are currently mutating these ATTA-like sequences and will determine if either VAX1 or

SIX3 are directly interacting with Per2 at these sites. Due to the close proximity of the

ATTA site at position +1782’s close-proximity to the VAX1-interacting site at position

+1774, it would not be surprising to see either VAX1 or SIX3 interaction at this site.

Regardless, our data suggest that VAX1 and SIX3 are both capable of modulating the molecular clock.

Other potential pathways through which SIX3 or VAX1 may influence circadian rhythms

Since we have not been able to establish exactly how SIX3 is altering circadian

35 rhythms in mice, it is possible that SIX3 may influence circadian rhythms via modulating other known homeodomain transcription factors which then target either SCN neuropeptide or molecular clock gene expression. Several other homeodomain transcription factors exist that are important for proper brain development and morphology. We have performed some preliminary experiments focused on two other homeodomain transcription factors, Six homeobox 6 (SIX6) and LIM homeobox 1

(LHX1), due to their abundant expression in the SCN during adulthood and requirement for SCN development (43,49,50). Preliminary transfection data have shown that Six6 and

Lhx1 overexpression modulates Per2-luciferase expression in NIH-3T3 mouse fibroblasts. The next step will be to establish if SIX3 can regulate the expression of these homeodomain transcription factors. We hypothesize that both SIX6 and LHX1 are required for the proper generation and output of circadian rhythms from the SCN, and that either could be downstream of SIX3.

Sex differences in circadian rhythms between male and female mice

Interestingly, our data showed that Vax1Syn:Per2Luc and Six3Syn:Per2Luc SCN explants had shorter and longer PER2::luciferase periods, respectively, in both males in females. However, this was not necessarily expected. VIP and AVP-expressing neurons project circadian information directly onto GnRH and Kisspeptin-releasing neurons in the hypothalamus, which form the start of the hypothalamic-pituitary-gonadal (HPG) axis, the primary hormonal negative feedback loop that regulates fertility and sexual development via release of sex steroid hormones: testosterone in males and estrogen and progesterone in females (51). It has been demonstrated that the sex steroid receptors are

36 expressed in neurons that compose the SCN, suggesting that these hormones may be capable of altering SCN function (52). Furthermore, our lab has recently been exploring how the female mouse estrous cycle can modulate circadian rhythms in the SCN.

Circadian rhythms have been demonstrated to alter the hypothalamic-pituitary-gonadal hormone axis (10). In line with the concept of the HPG axis being under the control of a hormonal feedback loop, treatment of mouse SCN explants with estrogen has shown to induce a longer PER2::luciferase period, and recent studies in our lab have suggested that estrogen surge prior to ovulation may be correlated with a lengthening in

PER2::luciferase (unpublished). Thus, even though we did not see any significant difference in PER2::luciferase period between male and female Vax1 or Six3 conditional knockout mice (female mice used were in metestrus), it is likely that sex differences may be exhibited if performed at different stages in the female estrous cycle.

In conclusion

We have identified a novel mechanism for modulation of circadian rhythms in the

SCN. Thus far, we know that VAX1 is required for SCN circadian rhythm cycling and output and that SIX3 is necessary to maintain proper circadian rhythms as well. With circadian biology becoming an ever-growing field in biomedical research as people are discovering that circadian rhythms play a role in the regulation of most of the systems in the living organism, further understanding of the molecular mechanisms behind the genesis, maintenance, and output of circadian rhythms will allow us to address an entire new level of regulation and development of biological systems. Shift-work, jetlag, time- changes, and artificial lighting are all issues that are becoming more and more present in

37 society, all of which have drastic effects on circadian rhythms and overall health.

Identifying new targets that modulate circadian rhythms will undoubtedly lead to several future possibilities for clinical targets for treating many of the common disorders that arise from abnormal circadian rhythms. Both VAX1 and SIX3 have been shown to be necessary for proper development and output of circadian rhythms in the SCN. With both the rise in daily activities that alter circadian rhythms and the rapid discovery of the circadian origin of many common diseases, the novel functions of VAX1 and SIX3 that we have discovered may someday play a pivotal role in the invention of cures and treatments for the several health crises that arise from a circadian origin.

38 References

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