Generation of Novel Conditional and Hypomorphic Alleles of the Smad2 Gene and the Effects of Smad2 Removal in Environments with Elevated Retinoid Signaling

GENERATION OF NOVEL CONDITIONAL AND HYPOMORPHIC ALLELES OF THE SMAD2 GENE AND THE EFFECTS OF SMAD2 REMOVAL IN ENVIRONMENTS WITH ELEVATED RETINOID SIGNALING

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Maria Helen Festing, B.S.

*****

The Ohio State University 2007

Dissertation Committee: Approved by Professor Michael Weinstein, Ph.D., Advisor

Professor Susan Cole, Ph.D. ______Professor Harold Fisk, Ph.D. Advisor Graduate Program in Molecular Genetics Professor Amanda Simcox, Ph.D.

ABSTRACT

Smad2 is an intracellular mediator of the transforming growth factor beta

(TGF-β) signaling pathway, and regulates target gene transcription. It has been

previously shown that, in the mouse, removal of functional Smad2 results in

embryonic lethality due to defects in gastrulation. To circumvent this early

lethality, the Cre-loxP system was utilized to generate a Smad2 conditional allele.

Cre-mediated recombination results in a deletion allele which phenocopies the previously reported Smad2ΔC null mutation. The hypomorphic allele Smad23loxP was also created that can be maintained as a homozygote. However,

Smad23loxP/Δ embryos are lethal and encompass a variety of phenotypes,

including craniofacial midline defects.

Holoprosencephaly (HPE) comprises a spectrum of craniofacial midline

defects and is a congenital cephalic disorder. Many candidate factors have been

identified that are associated with HPE in humans and animals, which include

excessive alcohol and retinoic acid exposure, and mutations or deletions at 12 genetic loci. Seven of these 12 loci have been identified, and include TGIF.

TGIF is a corepressor of the retinoid signaling pathway, and removal of this

ii repressor results in increased retinoid signaling. TGIF can also function in as a corepressor the transforming growth factor-beta (TGF-β) pathway, interacting with Smad2.

Research has indicated that mouse embryos harboring Tgif mutations are more susceptible to teratogenesis induced by exogenous all-trans retinoic acid

(atRA) when compared to wild-type littermates, and frequently display HPE.

Interestingly, embryos harboring either Smad2 null or hypomorphic alleles are also more susceptible to atRA teratogenesis, and frequently display HPE.

Embryos that harbor mutations in both Smad2 and Tgif exhibit a partially- penetrant craniofacial phenotype, which encompasses the HPE spectrum. This indicates that retinoid signaling may be involved in these HPE phenotypes.

There are many proteins involved in retinoid signaling, which includes both the retinoid signaling cascade and retinol metabolism. Analysis of gene transcription to determine a possible inherent susceptibility to teratogenesis in

Smad2 heterozygous embryos indicated that Adh4 and Cyp26A1, which are both involved in retinol metabolism, were reduced in early embryonic stages when forebrain development is beginning. These transcriptional reductions do not by themselves result in lethality, but when combined with any other factor that perturbs retinoid signaling, HPE can result. Finally, as another method to affect retinoid signaling, a Cyp26A1 null allele was bred to the Smad2 heterozygotes.

Consistent with affecting retinoid signaling, the Cyp26A1-/-; Smad2ΔE9,10/+ embryos display midline defects, including HPE.

iii

DEDICATION

To my mother Fran, my father Tom, my brother Tommy, and my grandparents Bill and Helen, for their support and patience over the years.

It is necessary to learn when to follow, and when to lead.

ACKNOWLEDGMENTS

I wish to thank my advisor, Dr. Michael Weinstein, for his advice and for allowing me to pursue the project designs, and Dr. Susan Cole, for helping with lab supplies.

I am grateful to all my committee members throughout my graduate

education: Dr. Susan Cole, Dr. Harold Fisk, Dr. Michael Ostrowski, and Dr.

Amanda Simcox for their encouragement.

I thank current and former members of the Weinstein Laboratory including

Dr. Tessa Carrel, Dr. Mark Hester, Dr. Ye Liu, Samuel Lasse, Kara Keplinger,

Samantha McCarthy, J Chris Thompson, Andy Chow, and Joe Mills for their

insight, help, and discussion over the years. I also wish to thank Dr. Heithem El-

Hodiri for his expertise in helping to perform the Xenopus laevis micro-injections.

Finally, I wish to thank the editors of Genesis for the use of the previously

published text and figures that are used in this dissertation. Generation of Novel

Conditional and Hypomorphic Alleles of the Smad2 Gene, 40:118-123, is

reprinted with the permission of Wiley-Liss, Inc., a subsidiary of John Wiley &

Sons, Inc.

VITA

30 March 1979………………… Born – Tacoma, WA.

May 2000………………………. The Pennsylvania State University, State College, PA. Biology (Genetics), BS.

2002 – present………………… Graduate Teaching Assistant. The Ohio State University.

PUBLICATIONS

Ye Liu, Maria Festing, John C Thompson, Mark Hester, Scott Rankin, Heithem M El-Hodiri, Aaron M Zorn, and Michael Weinstein. Smad2 and Smad3 coordinately regulate craniofacial and endodermal development. Dev Biol. 2004 June 15; 270(2): 411-426.

Ye Liu, Maria H Festing, Mark Hester, John C Thompson, and Michael Weinstein. Generation of novel conditional and hypomorphic alleles of the Smad2 gene. Genesis. 2004 Oct; 40(2):118-123.

FIELD OF STUDY

Major Field: Molecular Genetics

TABLE OF CONTENTS

Page

Abstract ……………………………………………………………………………….. ii

Dedication…………………………………………………………………………….. iv

Acknowledgments…………………………………………………………………… v

Vita…………………………………………………………………………………….. vi

List of Tables…………………………………………………………………………. x

List of Figures……………………………………………………………………….. xi

List of Abbreviations………………………………………………………………… xiii

Chapters:

1. Introduction……………………………………………………………………. 1 1.1 Introduction……………………………………………………………. 1 1.2 Development of the Early Mouse Embryo…………………………. 2 1.3 Neural Tube Development and Defects……………………………. 3 1.3.1 Neural Tube Formation and Neural Tube Defects.………. 3 1.3.2 Prosencephalic Development an Holoprosencephaly...… 4 1.4 TGF-β Superfamily Signaling…………...………………………...… 6 1.4.1 The Receptors…….……………………………………….…. 7 1.4.2 The Smads………….……………………………………...... 8 2. Generation of Smad2 Conditional and Hypomorphic Alleles…………….. 16 2.1 Introduction……………………………………………………………. 16 2.2 Results……………………………………………………………….... 17 2.2.1 Creation and Confirmation of the Smad2 Alleles…………. 17 2.2.2 Smad2 3loxP Allele is Hypomorphic……………………….. 21 2.2.3 Conditional Removal of Smad2…………………………….. 24 2.3 Conclusions………………………………………………….………... 26

vii 2.4 Materials…………….………………………………………………… 28 2.4.1 Generation of Targeted ES Cells and Creation of Germline Chimeric Mice….…………………………………. 28 2.4.2 Southern Blot………………………………………………..... 29 2.4.3 Mice and Matings…………………………………………….. 30 2.4.4 Genotyping……………………………………………………. 31 2.4.5 Histology………………………………………………………. 32 2.4.6 Hematoxylin and Eosin Staining…………………………… 33 2.4.7 Xenopus laevis Injection Assay…………………………….. 34 2.4.8 RNA Extraction……………………………………………….. 35 2.4.9 Real-Time PCR…………………………………………...….. 36 3. Removal of Smad2 Results in Increased Sensitivity to Retinoic Acid Induced Holoprosencephaly…………………………………………………. 50 3.1 Introduction…………….……………………………………………… 50 3.1.1 Retinoic Acid Metabolism…………………………………… 51 3.1.2 Retinoid Signaling……………………………………………. 52 3.1.3 Retinoid Signaling Affects TGF-β Signaling………………. 54 3.2 Results………………………………………………………………… 55 3.2.1 Retinoic Acid Signaling is Affected in Smad2 Embryos…. 55 3.2.2 Sensitivity of Smad2 Embryos to Retinoic Acid Teratogenesis………………………………………………… 57 3.2.3 Retinoic Acid Metabolizing Enzymes ADH4 and Cyp26A1 are Affected in Smad2 Embryos……….…………...……… 59 3.3 Conclusions………………………………………………………….. 62 3.4 Materials…………….………………………………………………… 64 3.4.1 Mice and Matings…………………………………………….. 64 3.4.2 Genotyping……………………………………………………. 64 3.4.3 Teratogenic Application of Retinoic Acid………………….. 65 3.4.4 Whole Mount Visualization of β-galactosidase Expression……………………………………………………. 65 3.4.5 Quantification of β-galactosidase expression…………….. 66 3.4.6 RNA Extraction and Analysis……………………………….. 67 3.4.7 Real-Time PCR………………………...…………………….. 68 4. Smad2 and Tgif, in Addition to Strain Background, Work in Concert to Cause Murine Holoprosencephaly 80 4.1 Introduction….………………………………………………………… 80 4.2 Results………………………………………………………………… 80 4.2.1 Expression Domain of Tgif and Tgif2………………………. 80 4.2.2 Retinoid Signaling Affected in Tgif Embryos…………….... 83 4.2.3 Frequency and Penetrance of Holoprosencephaly in 84 Smad2/Tgif Embryos………………………………………… 4.2.4 Molecular Analysis of Smad2/Tgif Embryos………………. 87 4.2.5 Sensitivity of Smad2/Tgif Embryos to Retinoic Acid……... 89 4.3 Conclusions…………………...………………………………………. 90 4.4 Materials……………….……………………………………………… 94

viii 4.4.1 Mice and Matings…………………………………………….. 94 4.4.2 Genotyping……………………………………………….…… 95 4.4.3 Histology……………………………………………….……… 95 4.4.4 Whole Mount In Situ Hybridization………………….……… 96 4.4.5 Teratogenic Application of Retinoic Acid………….………. 99 4.4.6 RNA Extraction and Analysis……………………….………. 100 5. Smad2 and Cyp26A1 Mutations Work in Concert to Cause Murine Holoprosencephaly..…………..……………………………………………… 113 5.1 Introduction……………………………………………………………. 113 5.2 Results……………………………………………………...……….… 115 5.2.1 Frequency and Penetrance of Holoprosencephaly in 115 Smad2/Cyp26A1 Embryos….………………………………. 5.2.2 Cyp26A1 and the Smad2 Hypomorphic Allele…………… 117 5.2.3 Smad2, Cyp26A1, and Tgif………………….…………….... 117 5.3 Conclusions…………………………………………………………… 119 5.4 Materials...…………………………………………………………….. 120 5.4.1 Mice and Matings……………………..……………………… 120 5.4.2 Genotyping…………………..……………………………….. 120 6. Discussion…………………………………………………………...………… 126 6.1 Smad2 Alleles and Phenotypes…..………………………………….. 126 6.2 Smad2, Retinoid Signaling, and HPE………..……………………… 130 Bibliography……………………………………………………………………….. 137

LIST OF TABLES

Table Page

2.1 Heterozygote interbreeding of the Smad2 alleles…………………. 38

2.2 Primer sequences………….………………………………………….. 39

2.3 Intercrosses of the conditional alleles to the null allele……………. 40

2.4 Intercrosses of Smad2flox/flox to various Cre mouse lines…………. 41

3.1 Primer sequences……………………………………………………... 69

4.1 Intercrosses between Smad2 and Tgif result in partially- 101 penetrant HPE………………………………………………………….

4.2 Primer sequences……………………………………………………... 102

5.1 Intercrosses between Smad2/Cyp26A1 and Cyp26A1 result in 122 partially-penetrant HPE………………………………………………..

5.2 Intercrosses between Smad2, Cyp26A1, and Tgif………………… 123

5.3 Primer sequences……………………………………………………... 124

LIST OF FIGURES

Figure Page

1.1 Development of the early mouse embryo……………………………. 11

1.2 Mammalian neutralization……………………………………………… 12

1.3 Defects in prosencephalon cleavage…………………………………. 13

1.4 TGF-β signaling cascade…………………………………………. ….. 14

1.5 Relationship and structure of the mammalian Smads……………… 15

2.1 Generation of the Smad23loxP allele and mouse…………………….. 42

2.2 Alleles from Cre-mediated recombination of Smad23loxp and the PCR strategy used to identify them………..………………………..... 43

2.3 Protein sequence of various Smad2 products…………………...... 44

2.4 Analysis of Smad2ΔE9,10 allele……………………………………...….. 45

2.5 Analysis of the Smad2ΔE9,10 allele compared to dominant-negative Smad2 constructs………………………………………………………. 46

2.6 Smad23loxP/ΔE9,10 embryos are early embryonic lethal………………. 47

2.7 Aberrant splicing of the Smad23loxP allele……………………………. 48

2.8 Real-time PCR of wild-type Smad2 transcript levels found in various Smad2 genotypes………………….………………………….. 49

3.1 Retinoic acid synthesis and degradation…………………………….. 70

3.2 Retinoid signaling…………………………………………………….…. 71

3.3 Retinoid signaling level in Smad2ΔE9,10/+ and wild-type embryos….. 72

3.4 Retinoic acid induces HPE in Smad2 embryos……………………... 74

3.5 Expression level of RARs and RXRs…………………………..…….. 75

3.6 Expression level of RA metabolizing genes……………………...….. 76

3.7 Real-time PCR of ADH4 in Smad2 embryos…………………...……. 77

3.8 ADH4 promoter sequence………………………………………...…… 78

3.9 Cyp26A1 promoter sequence………………………………………..... 79

4.1 Expression of Tgif in early-stage mouse embryos………………..… 103

4.2 Expression of Tgif2 during mouse embryogenesis…………………. 105

4.3 Retinoid signaling level in Tgif mutant embryos…………………….. 106

4.4 Holoprosencephaly phenotype………………………………………... 107

4.5 Breeding scheme for Smad2/Tgif mutant mice…………………….... 108

4.6 Holoprosencephaly phenotype………………………………………... 109

4.7 Affects of Smad2 on a near-congenic C57BL/6 background………. 110

4.8 Expression level of RA metabolizing genes…………………………. 111

4.9 Severity of phenotype observed in Smad2/Tgif embryos exposed to atRA…………………………………………………………………… 112

5.1 Holoprosencephaly phenotype………………………………………... 125

6.1 Model of TGF-β and retinoid signaling pathways…………………… 136

xii

LIST OF ABBREVIATIONS

+ positive (number) or wild-type allele (genotype)

- negative (number) or null allele (genotype)

% percent

female

male

α alpha

ADH4 Alcohol dehydrogenase class IV (ADH7) atRA all trans retinoic acid

β beta

BMP bone Morphogenetic protein

°C degrees Celsius

C Carbon atom

C-terminal carboxyl-terminal cDNA complementary DNA

CNS central nervous system

CO2 carbon dioxide

Δ deletion

xiii d distilled

d2 double distilled

DepC diethylpyrocarbonate

DMF N,N-dimethylformamide

DMSO dimethylsulfoxide

DNA deoxyribonucleic acid

E embryonic day (embryo) or exon (allele)

ES Mouse embryonic stem cells

EtBr ethidium bromide

EtOH ethanol

Fig. Figure

γ gamma g gram(s) h human

H hydrogen ion

H2O water

HAT histone acetyl transferase

HDAC histone deacetylace

HPE holoprosencephaly

hr hour(s)

k kilo

kDa kilodaltons

L liter(s)

xiv LPM lateral plate mesoderm

µ micro m meter

M moles per liter

MeOH methanol

MH mad homology min minute(s) mM milliMole mol mole(s) mRNA messenger RNA n number of samples neo neomycin ng nanogram(s)

NTD neural tube defect

O Oxygen atom

OH hydroxyl group

P Postnatal day

PBS phosphate buffered saline

PCR polymerase chain reaction

PFA paraformaldehyde

+ pH -log10 [H ]

RA retinoic acid

RAR retinoic acid receptor

xv RARE RA response element

RNA ribonucleic acid

RT reverse transcriptase

RXR retinoid X receptor

RXRE RXR response element

SARA Smad Anchor for Receptor Activation

SBE Smad Binding Element

Shh Sonic Hedgehog

Sm Smad

Smad Sma (small) and Mad (mothers against decapentaplegic)-related

protein

Smad2 refers to both Smad2 isoforms

TBS tris buffered saline

TGF-β Transforming Growth Factor-beta; references ligand, pathway, and

the superfamily

TGIF 5’ TG 3’ interacting factor

UV ultraviolet

WT wild-type; non-transgenic allele

xvi

CHAPTER 1

INTRODUCTION

1.1 Introduction

Due to similarity between humans and other organisms, it is possible to use these other organisms to study the processes that occur in humans. To understand what occurs in early development and craniofacial patterning, the murine model is ideal, since there is a high degree of homology between human and mouse. It is this similarity, and the ability to perform complex genetic techniques and manipulations, that make the mouse model ideal for studying these early processes. The purpose of this study was to investigate mouse embryos harboring mutations in Smad2 alone, and in combination with mutations in genes associated with retinoid signaling and metabolism, in order to elucidate the function and role of Smad2 in murine early development and craniofacial formation. Novel mechanisms have been uncovered, and new Smad2 alleles have been created that will assist in further defining the role of Smad2 in many other developmental processes.

1 1.2 Development of the Early Mouse Embryo

Early mouse development can be reviewed in [1, 2]. At the time of

implantation, the embryo consists of a small sphere of cells termed the inner cell mass, surrounded by an outer layer of trophoblast cells. Early post-implantation

mouse embryos consist of three types of tissue, the extra-embryonic ectoderm

(ExE) from the trophoblasts, the visceral endoderm (VE), and the epiblast (Fig.

1.1). The ExE and VE are extra-embryonic, and will not contribute to the embryo

proper. All the tissues of the embryo will derive from the epiblast. The first physical sign of patterning within the embryo occurs at E6.5, when gastrulation begins. A portion of the epithelium undergoes an epithelium-mesenchymal transformation and forms the primitive streak, the posterior pole of the embryo.

This primitive streak will elongate over the next 24 hrs, and at the most anterior

end, the node forms. This organizing center will generate axial midline

mesoderm, which will comprise the prechordal plate, notochord, and definitive

gut endoderm. However, before physical signs of patterning are evident,

molecular patterning has already taken place. Almost 12 hours before the

primitive streak will form, differential gene expression of Otx2, Lim1, goosecoid,

and Hex have been observed in the anterior visceral endoderm (AVE). The

anterior visceral endoderm is derived from select cells in the VE which express

Hex.

The AVE will migrate to the future anterior of the embryo. There these

cells function to induce the ectoderm to form anterior neural and non-neural

ectoderm, which is critical to correct anterior patterning. Removal of the AVE in

2 mouse before gastrulation occurs resulted in reduction in forebrain markers [3].

Removal of Lim1 also resulted in truncation of anterior head structures [4], and

removal of Hex resulted in anterior truncation [5].

1.3 Neural Tube Development and Defects

Mouse and human neural tube development is very similar; thus, studying

the mouse will aid in understanding the normal and abnormal process in humans

(reviewed in [6, 7, 8, 9]). Abnormal neural tube development can result from

genetic mutations or environmental factors.

1.3.1 Neural Tube Formation and Neural Tube Defects

Neuralization of the regions that later give rise to the brain and spinal cord

above the lumbar region is referred to as primary neuralization. Secondary

neuralization is associated with the caudal, or posterior, segments of the spinal

cord. During primary neutralization, the neural plate is formed from the ectoderm

above the notochord and chordal mesoderm, which releases inductive signals

like Follistatin and Noggin. The lateral margins of the plate invaginate and fuse

over a period of two days (in humans) to form the neural tube (Fig. 1.2A). Fusion

initiates at three sites, the cervical-hindbrain boundary, the forebrain-midbrain

boundary, and the rostral extremity, or most anterior portion, of the forebrain.

Closure continues from these sites to completion sites termed neuropores. From

this tube the central nervous system (CNS) will arise. Primary neutralization

occurs in the 3rd week of human gestation and E8.0-9.0 for the mouse.

3 Differentiation of cells in the ventral neural tube and induction of the formation of

the floor plate is induced by SHH, which is secreted from the notochord. The

anterior portion of the neural tube will form the prosencephalon.

Disturbances in this early patterning process result in some of the most severe neural tube defects of the CNS, with many cases spontaneously aborting.

Total lack of neutralization results in craniorachischisis totalis, while failure of

neural tube closure results in two disorders depending on which part of the

neural tube fails to close – anterior failure leads to anencephaly, while posterior

failure leads to myeloschisis (reviewed in [6, 7, 8, 9]).

1.3.2 Prosencephalic Development and Holoprosencephaly

Development of the prosencephalon occurs in three phases.

Prosencephalic formation occurs at the anterior portion of the neural tube after

the anterior neuropore closes. The second phase, prosencephalic cleavage,

involves horizontal, transverse, and sagittal cleavage of the neural tube.

Horizontal cleavage will form the optic vesicles and olfactory bulbs, transverse

cleavage will separate the telencephalon from the diencephalon, and sagittal

cleavage of the telencephalon into the left and right hemispheres and lateral

ventricles (Fig. 1.2B). This occurs early in development- the 5th week for

humans, and E9.0-10.0 for the mouse. Finally, the structures that will form the

corpus callosum, septum pellucidum, optic nerve chiasm, and hypothalamus

4 begin to develop during the midline prosencephalic development phase. When

these stages of development are altered, or the separation is not complete, the prosencephalic disorder holoprosencephaly (HPE) can result.

Holoprosencephaly is the most common neural tube defect in humans,

affecting 1 out of every 250 conceptions [10]. However, due to the high rate of

lethality associated with this disorder, it is only observed 1 out of every 16, 000

live births [11]. HPE is a spectrum disorder, encompassing many different severities of the prosencephalic region. The most severe HPEs include aprosencephaly, pseudoholoprosencephaly, and atelencephaly, though they are sometimes classified as separate disorders from HPE due to the lack of an initial complete prosencephalic structure. When those disorders (aprosencephaly, pseudoholoprosencephaly, and atelencephaly) are classified separately, HPE then refers to when the prosencephalic structure failed to undergo the three cleavages completely. Regardless of whether to define HPE as including aprosencephaly, pseudoholoprosencephaly, and atelencephaly or not, these severe forms tend to abort early in gestation, while the other HPE forms develop further. These milder forms of HPE are divided into three main categories (Fig.

1.3). The most severe, alobar, display no hemispheric division, and can be

subdivided into the pancake type, the cup type, and the ball type, depending on

the degree of membranous ventricular roof coverage that is present. Semilobar

has varying extent of division, while lobar is the mildest, with almost complete

hemispheric division. Disorders that are associated with HPE include the

absence of the corpus callosum and arhinencephaly (reviewed in [6, 7, 8, and

5 9]). A variety of mutant alleles are associated with mammalian cases of HPE as either causative or contributing factors to this disorder [12]. These include mutations in SHH [13], TGIF [14], and PATCHED [15]. Additionally, environmental causes have been associated with HPE and can include RA [16], alcohol [17], and cyclopamine which can be found in Veratrum californicum [18].

1.4 TGF-β Superfamily Signaling

Transforming Growth Factor-beta (TGF-β) is a major signaling cascade, and is involved in almost every aspect of development and growth. TGF-β signaling has been observed in proliferation, apoptosis, differentiation, gastrulation, and patterning of the embryo axis [19]. TGF-β is also associated with disease progression and some forms of cancer. The TGF-β superfamily encompasses both the TGF-β and Bone Morphogenetic Protein (BMP) signaling cascades; both signaling pathways are present in every cell (reviewed in [20, 21

22, 23, 24, 25]). Over 40 signaling ligands are found in mammals, and include

TGF-βs, BMPs, anti-Müllerian hormone, nodal, and activins. The ligand is translated as a prohormone that is later cleaved to release the mature C-terminal fragment. This fragment will remain non-covalently associated with the N- terminal fragment (LAP [latency-associated peptide]) after secretion; this complex is termed the small latent complex (SLC). The SLC associates with latent TGF-β binding proteins (LTBP), to form the large latent complex (LLC).

The LLC is present in the extracellular matrix, but is not recognized by the signaling receptors. Ligand half-life in the extracellular matrix is short, but is

6 significantly increased by being associated with the LLC [26]. The ligand is

active when released from the LAP; this separation can be mediated by the

integrins α8β1 [27], αvβ6 [28], and αvβ8 [29].

1.4.1 The Receptors

Active ligands will bind to a serine/threonine protein kinase receptors

located on the cell surface. Diffusible ligand-binding proteins are present that,

when bound to the ligand, prevent the ligand access to these receptors. Ligand-

binding protiens include the binding proteins Cerberus, Chordin, or Follistatin

(Fig. 1.4). Ligands successfully bypassing these ligand-binding proteins will

complex to the receptors. In mammals, there are seven Type I receptors and

five Type II receptors for both TGF-β and BMP signaling. The ligand associates

with the Type II receptor; this receptor will then become activated and

homodimerizes with another Type II receptor. This complex will then

heterodimerizes with two Type I receptors. The Type II receptor will

phosphorylate the GS region (an intracellular portion of the receptor containing

the SGSGSG motif) of the Type I receptor, resulting in the activation of the Type I

receptors’ kinase domain. The Type I receptors will in turn phosphorylate only

the intracellular mediators, the Smad proteins, at the C-terminal serines (an

SSxS motif). There are a variety of methods to regulate and control TGF-β signaling at the receptors. Accessory receptors exist that facilitate the binding of the ligand to the signaling receptors and include betaglycan/Type III TGF-β receptor. Factors also exist that inhibit receptor function and include FKBP12

7 and FKB12.6, which bind directly to the GS domain in Type I receptors, and are thought to prevent leaky activation that may result from random encounters with the Type II receptor that has no bound ligand. The receptor decoy BAMBI (BMP and activin membrane-bound inhibitor) can heterodimerize with Type I receptors through the E6 loop, interfering and preventing BMP signaling.

1.4.2 The Smads

The Smad Family of effector proteins are inter-related (Fig. 1.5 A); Smad2 and Smad3 are activated by TGF-β ligands, while Smads 1, 5, and 8 are

activated by BMP ligands. These regulated Smads are known as R-Smads, and

have three main domains. A Mad Homology (MH) 1 domain, which is involved in

DNA binding through a β hairpin loop, a non-conserved linker region where there

are sites for MAPK phosphorylation, and the C-terminal MH2 domain involved in

protein: protein interactions and binding to cofactors, which occurs in the α helix-

2; this domain is also where the serines are located for activation (Fig. 1.5 B).

Receptor specificity of the Smad proteins to the respective Type I receptor is achieved through the L3 loop and the α helix-1 in the MH2 domain and the L45 loop in the kinase domain of the receptor. The Smad proteins are also regulated in their ability to access the receptor, be phosphorylated, and enter the nucleus.

Before activation, an individual R-Smad is bound to itself, the MH2 domain covering the nuclear localization signal (NLS) located in the MH1 domain. To facilitate phosphorylation from the activated receptor, some Smad2 and Smad3 are bound to SARA via an asparagine located in the MH2 domain

8 [30]. The C-terminal domain of SARA can interact with the activated receptor

complex, thus localizing Smad2 and Smad3 close to the receptors to be

phosphorylated, increasing phosphorylation efficiency. Unphosphorylated

Smad2 and Smad3 not associated with SARA is sequestered in the microtubule

cytoskeleton [31]. The BMP Smads 1 and 5 can be regulated by Smurf-1, which

results in ubiquitination and degradation, independent of Smad activation.

Upon phosphorylation, the Smads disassociate from SARA, and associates with Smad4, the common Smad (Co-Smad). The structure of Smad4

is similar to the R-Smads, but does not contain the NLS and C-terminal serines.

Smad4 has the ability to bind to DNA, but this binding is the weakest of all the

Smads. This R-Smad-Smad4 complex translocates into the nucleus where it binds to Smad Binding Elements (SBE), which are tandem repeats of the simple sequence CAGA, via a DNA binding domain (DBD) located in the MH1 domain

[32, 33]. Two isoforms exist of Smad2, one in which the DBD is present (this protein is referred to as Smad2ΔE3) [34], while the other isoform contains exon 3,

which results in a disruption in the beta helix of the DBD and abolishes binding

ability (this protein is referred to as Smad2FL) [35]. It was previously thought that

only 10% of total Smad2 protein was Smad2ΔE3, but this result has been

challenged, and Smad2ΔE3 may represent the majority of Smad2 protein present

in a cell [34]. Alternative splicing that affects the function, and thus level of TGF-

β signaling, is not unique; Smad4 has six isoforms, while only three are

associated with increased TGF-β signaling [36]. Interaction with coactivators and

corepressors assist in enhancing or repressing gene transcription; these

9 cofactors include TGIF, p300/CBP, c-Ski, Vitamin D receptor, Milk, and Mixer.

These coactivators and corepressors modify the chromatin around the target gene via recruitment of HATs and HDACs. TGIF, in addition to functioning as a corepressor with Smad2, can inhibit the phosphorylation of Smad2 [37], and function as a corepressor of retinoid signaling [14].

TGF-β signaling can also be inhibited by Smads 6 and 7 (I-Smads), which contain the three domain of the Smads, but are very divergent from the other

Smads. Smad6 preferentially inhibits BMP signaling [38] and does so by binding to activated Smad1, in place of Smad4. This results in an ineffective Smad complex. At high expression levels, Smad6 can bind to the Type I receptors and inhibit TGF-β and BMP signaling [39]. Smad7 preferentially inhibits TGF-β signaling, and does so by interacting with the Type I receptors [38]. Additionally, dephosphorylation of the Smad proteins can occur. Recently, is was observed that PPM1A dephosphorylates Smad2 and Smad3, promoting nuclear export and inhibiting TGF-β signaling [40]. All of these enhancing and repressing processes help to attenuate TGF-β signaling in the developing mammal.

Figure 1.1 Development of the early mouse embryo. Graphical representation of tissue and cell movement during post-implantation and gastrulation of E5.5-7.0 mouse embryos. From [1].

Figure 1.2 Mammalian neuralization. A. Primary neutralization is a multi-step process. The neural plate begins to invaginate (shaping and folding), and then the sides of the neural plate and neural crest begin to converge and fuse (elevation and convergence). During closure the neural plate uses into a cylindrical structure called the neural tube. This process occurs during gestation at stages E8.0-9.0 in mice, and day 24-26 in humans. Taken from [41]. B. Mammalian neural tube development. The anterior portion of the neural tube, the prosencephalon, will cleave to form the left and right hemispheres of the telencephalon, diencephalon, and the ventricles. This process occurs during gestation at stages E9.0-10.0 in mice, and week 5-6 in humans. Altered from [42].

Figure 1.3 Defects in prosencephalon cleavage. Upper panel illustrates the relationship between increasing severity of HPE and reduction of hemispheric division. Lower panel shows the three categories of HPE abnormalities that can be observed. Lobar, the mildest, display almost complete division along the midline (in this case there is only a single front tooth). Semi-lobar have varying degrees of separation, while alobar has almost no separation. Note the single eye field and proboscis above. Adapted from [43].

Figure 1.4 TGF-β signaling cascade. Extracellular ligand successfully bypassing ligand-binding proteins bind to the Type II receptor. Type II receptor homodimerizes, then this complex heterodimerizes with two Type I receptors, and phosphorylation occurs to activate the Type I receptor. Smad 2 and Smad3 are bound to themselves, and to SARA, covering the nuclear localization signal and positioning the Smads close to the receptors. Type I receptor phosphorylates the Smads at the C-terminal serines. Upon activation, the Smads change conformation, release from SARA, complex with Smad4, and translocate into the nucleus. Interaction with transcription cofactors, coactivators, and corepressors result in target gene activation or repression. BMP signaling is similar, but involving BMP ligands, BMP receptors, and Smads 1, 5, and 8. Altered from [41].

Figure 1.5 Relationship and structure of the mammalian Smads. A. Smad phylogenetic tree. This tree displays the relationship between the mammalian R- Smads, Co-Smad, and I-Smads. Note that the Smads responsive to BMP signaling cluster together, while TGF-β responsive Smads cluster together. The tree was generated using ClustalX [44]. B. General Smad protein structure. Smads all possess an MH1, linker, and MH2 domains. Smads 1, 2ΔE3, 3, 5, and 8 all contain a nuclear localization signal (NLS) and DNA binding domain (DBD) in the MH1 domain. Smad2FL isoform contains exon3, which affects the DBD conformation, rendering it nonfunctional. All R-Smads contain an SSxS motif in the C-terminal portion of the MH2 domain, important for activation, and an L3 loop (L3) which interacts with the Type I receptor. Smad4 is similar to the R- Smads, but lacks the NLS and SSxS motif. Smads 6 and 7 are dissimilar to the other Smad proteins, especially in the MH1 and MH2 domains, but do contain the MH core sequence, so are classified as Smads.

CHAPTER 2

GENERATION OF SMAD2 CONDITIONAL AND HYPOMORPHIC ALLELES

2.1 Introduction

Smad2 and Smad3 are the intracellular transmitters for TGF-β, Nodal, and

Activin signaling ligands [45, 46]. Upon ligand binding, the receptors are

activated, and in turn activate Smad2 and Smad3 via phosphorylation of the C-

terminal serine residues in the MH2 domain. These activated mediators then

bind with Smad4 and subsequently translocate to the nucleus. Once in the

nucleus this Smad complex, through interactions with other transcription factors, regulates the expression of responsive genes [47, 48, 49, 50]. To determine the role and function of Smad2 during mammalian development, distinct loss of function alleles have been previously generated. Each of these Smad2 alleles result in early embryonic lethal phenotypes when null [51, 52, 53, 54, 55, 56], though the phenotypes can be different depending on the allele used.

Smad2Robm1/Robm1 embryos display an empty yolk sac phenotype that has

disorganized polarity and contains mesoderm [55]. This is in contrast to

Smad2ΔC/ΔC embryos, which display severe gastrulation defects and lack

mesoderm [56].

16 In an effort to circumvent the early lethality and analyze Smad2 function in stages beyond gastrulation, the Cre-loxP system had been adopted to create and use Smad2 conditional alleles. Cre is a bacteriophage P1-derived recombinase which excises the DNA between two loxP recognition sites in the same orientation [57]. A previously published Smad2 conditional allele was designed

to remove the first coding exon; thus with no start codon, no protein would be

translated [58]. However, a truncated Smad2 protein had been found in cells

homozygous for a similar Smad2 mutant allele which lacks the first coding exon

[52]; this truncated protein can be phosphorylated and translocate into the

nucleus [59]. Thus, this fragment may still have some residual function.

Therefore, a novel conditional allele was designed that, in the presence of Cre

recombinase, will result in a recombined allele where exons 9 and 10 of the MH2

domain are removed. Thus, if a protein is created, it will lack the majority of the

MH2 domain, including the C-terminal phosphorylation sites which are needed

for activation of Smad2.

2.2 Results

2.2.1 Creation and Confirmation of the Smad2 Alleles

A targeting construct used to create the Smad23loxP allele (named for the 3

loxP sites) was generated by placing a loxP site into intron 8 of the Smad2 wild-

type genomic sequence, and a floxed PGKneo cassette [60] was placed into

intron 10 (Fig. 2.1A). This construct was electroporated into TC1 ES cells [60]

and after G418 and gancyclovir double selection, 10 homologous recombinants

17 were identified by Southern blot analysis using a 5’ external probe (Fig. 2.1B).

Injection of targeted ES cells into C57BL/6 blastocysts generated chimeric mice,

and when crossed to Black Swiss mice, resulted in germline transmission of the

Smad23loxP allele. Mice heterozygous for the Smad23loxP allele were viable, healthy, and fertile. Heterozygous mice were intercrossed and wild-type, heterozygous, and homozygous Smad23loxP mice were generated at normal

Mendelian ratios (Table 2.1), and genotypes were confirmed by Southern blot analysis using a 5’ external probe (Fig. 2.1C). The homozygotes were viable, healthy, and fertile, and are indistinguishable from their heterozygous and wild- type littermates. Mice harboring a Smad23loxP allele were then mated with the

EIIa-Cre transgenic mice line [61] to create germline recombination between the

three loxP sites. This breeding generated all possible recombined alleles in a

mosaic mouse as follows: recombination between loxP1 and loxP2, deleting

exons 9 and 10 only (Smad2neoΔE9,10); between loxP2 and loxP3, removing the neo cassette only (Smad2flox); and between loxP1 and loxP3, excising exons 9,

10, and the neo cassette (Smad2ΔE9,10) as diagramed in Figure 2.2. The latter

deletion introduces a shift in the reading frame of exon 11, which encodes part of

the MH2 domain, resulting in an early stop codon and a predicted truncated

protein lacking the L3 loop and having no function (Fig. 2.3). The mosaic parent

was bred to a wild-type mouse for isolation of each of the recombined alleles,

which were identified by PCR analysis using the various sets of indicated primers

(Fig. 2.2 and Table 2.2 and data not shown). Smad2flox/+ and Smad2flox/flox mice

18 were produced at Mendelian ratios (Table 2.1) and were healthy and fertile. The

generation of Smad2neoΔE9,10/+ and Smad2neoΔE9,10/neoΔE9,10 mice was not pursued.

Previous work with another allele that has a deletion of the MH2 domain in

Smad2 (Smad2ΔC) resulted in recessive lethality before embryonic day 8.5

(E8.5). Smad2ΔC/ΔC embryos observed at E6.5 were smaller than wild-type and lacked the extraembryonic portion of the egg cylinder [56]. In order to verify that the Smad2ΔE9,10 allele behaved in a similar fashion to Smad2ΔC, Smad2ΔE9,10/+ mice were intercrossed and homozygous Smad2ΔE9,10 embryos were analyzed,

as no Smad2ΔE9,10/ΔE9,10 mice were born (Table 2.1). These null embryos were

observed to be much smaller than their wild-type littermates at E7.5 (Fig. 2.4Ai

and iii), and appeared indistinguishable from the Smad2ΔC/ΔC embryos [56].

Histological sections of wild-type and null embryos at E6.5 reveal that the null

embryos are small and lack discernable structures (Fig. 2.4B). Many

researchers have occasionally observed heterozygous Smad2 mutant embryos

that are external to the yolk sac [53, 62] which were also observed in less than

5% of the Smad2ΔE9,10/+ embryos (Fig. 2.4Aii).

There has been controversy surrounding the various Smad2 deletion

alleles, which can display different phenotypes, though all are early embryonic

lethal [51, 52, 53, 54, 55, 56]. This variation in observed phenotype among the

different Smad2 alleles may be partially due to the deletion of different regions of

the Smad2 gene. Thus, any protein that may be produced could have some

function, depending upon the domains not manipulated. Generally, N-terminal

mutations of Smad2 result in milder homozygous phenotypes than C-terminal

19 mutations like those produced with the Smad2ΔC and Smad2ΔE9,10 alleles [52, 55,

56, 58]. Alternatively, it is possible that the Smad2ΔC, and by extension

Smad2ΔE9,10, mutation created a Smad2 protein with antimorphic effects upon

TGF-β signal transduction [63]. In order to address this alternative possibility, a

Smad2 cDNA corresponding as closely as possible to the mRNA transcript of the

Smad2ΔE9,10 allele (hSmad2ΔE9-11) was assayed for potential dominant-negative activity in a Xenopus laevis embryo injection assay. The 32 uninjected and 19

LacZ injected embryos developed normally (Fig. 2.5A), with a dorso-anterior

index (DAI) [64] score of 5.0±0.0, as compared to injection of a known dominant-

negative form of Smad2, Smad23SÆA [63] (n=15), which exhibited anterior

truncation (Fig. 2.5B and C) and a DAI score of 4.67±0.7. However, no effects

were observed in the development of the Xenopus embryos injected with

hSmad2ΔE9-11 mRNA (Fig. 2.5D-F), as all 54 embryos injected exhibited a DAI

score of 5.0±0.0, despite the presence of a truncated Smad2 protein (Fig. 2.5J).

Therefore, deletion of exons 9 and 10 is most likely to create a null allele of

Smad2, and the observed phenotype in the Smad2ΔE9,10/ΔE9,10 embryos is not a

result of an antimorphic effect. This is in contrast to the slight anterior truncations

observed when a Smad2 construct lacking only the phosphoralytable serines is

injected (hSmad2ΔCTD) (Fig. 2.5G-I), which does produce a very mild antimorphic

effect. This construct is almost identical to one made by Lo, et al. [65] termed

Smad2(1-456). In vitro, this construct interacted with the receptor with greater efficiency than wild-type Smad2, and it was found that this interaction was dependant on the L3 loop in the MH2 domain. Thus, the antimorphic effect of

20 hSmad2ΔCTD is almost certainly due to the resulting protein having almost all of the MH2 domain present and thus, being able to interact with the receptors via

the L3 loop, but unable to be released from the receptors due to the lack of the

phosphorylation sites. The antimorphic effect may not be as strong as what was

observed with the Smad23SÆA construct possibly due to the difference of complete lack of the extreme C-terminus (hSmad2ΔCTD), as opposed to amino

acid substitutions while still maintaining the extreme C-terminus (Smad23SÆA).

The Smad2 alleles ΔC, ΔE9,10, and ΔE9-11 all lack the L3 loop, which is most likely the reason why these alleles are null. All Smad2 alleles where the N- terminus has been removed could still produce a protein containing an intact

MH2 domain, and thus, an intact L3 loop.

2.2.2 Smad2 3loxP Allele is Hypomorphic

It has been reported that the neo cassette contains cryptic splice donor and acceptor sites; therefore, insertion of a neo cassette intronically can result in a hypomorphic allele [66]. However, homozygous Smad23loxP mice are obtained at near Mendelian ratios (Table 2.1), and show no observable physical abnormality. In order to more completely characterize the Smad23loxP allele,

Smad23loxP mutant mice were bred with Smad2ΔC/+ mice. From these crosses, no

Smad23loxP/ΔC offspring were found, suggesting that this genetic combination is

lethal (Table 2.3A). The Smad2ΔE9,10 mutant allele was also bred to Smad23loxP,

resulting in the same apparent lethality, with no Smad23loxP/ΔE9,10 animals being

found in the 138 offspring analyzed (Table 2.3B). The timing of lethality was

21 determined through crosses of Smad2ΔE9,10/+ with Smad23loxP/3loxP animals. All of

the Smad23loxP/ΔE9,10 embryos were recovered as late as E10.5, and exhibited

lethality before E11.5 (Table 2.3C and data not shown). Between E8.5 and

E10.5 they displayed a range of phenotypes (Fig. 2.6), which can be divided into

three general classes, all of which could be observed in a single litter. Class I

mutants (Fig. 2.6A), which occur at a 30% penetrance, consist of an empty yolk

sac, phenocopying Smad2Robm1/Robm1, Smad2m1Mag/m1Mag, and Smad2m1Mag/Robm1 embryos [55, 67]. Class II mutant embryos (Fig. 2.6B), which are displayed 41% of the time, have a mass of tissue attached to the distal tip of the yolk sac and, in some cases, this tissue is large enough to display obvious anterior/posterior polarity. Finally, 23% of the embryos are Class III (Fig. 2.6C), which consists of embryos with severe midline defects as compared to their normal siblings (Fig.

2.6D). The phenotypic variability observed in theSmad23loxP/ΔE9,10 embryos is due

to the outbred nature of the genetic background (see section 5.2.3). To

determine whether this hypomorphic effect was due to the presence of the

intronic neo cassette, or an artifact caused by the insertion of the loxP sites,

breeding was established to produce Smad2flox/ΔE9,10 embryos. These embryos were found at normal Mendelian ratios, and are indistinguishable from littermates

(Table 2.3D). The embryonic lethality and the developmental abnormalities observed only in Smad23loxP/ΔC and Smad23loxP/ΔE9,10 embryos support a

conclusion that the Smad23loxP allele is a novel hypomorph, due to the affect of the neo cassette. The Smad23loxP allele was able to be maintained in the

homozygous state, and exhibited no observable anomaly until it was placed

22 opposite of a null allele. This was a milder hypomorphic allele than the

previously reported Smad2m1Mag allele [66], since Smad23loxP/3loxP mice are viable,

while Smad2m1Mag/m1Mag embryos were not.

To confirm that the Smad23loxP allele aberrantly spliced neo into the

Smad2 mRNA transcript (Fig. 2.7A), RNA was extracted from individual wild-

type, Smad23loxP/+, and Smad23loxP/3loxP E10.5 embryos and cDNA made. Non-

quantitative RT-PCR was done using primers specifically designed to identify a

Smad2-neo fusion transcript. It was observed that in only embryos which had at least one Smad23loxP allele could the fusion PCR product be detected (Fig. 2.7B).

Upon sequencing this product (data not shown), it was confirmed that neo is

aberrantly spliced into the Smad2 transcript, and this fusion between exons 10

and 11 would result in a truncated product due to the introduction of a stop codon

encoded by neo, which is out of the normal reading-frame. Thus, any Smad2-

neo protein produced would not function as a wild-type protein, due to the lack of

a portion of the MH2 domain, which would include the SSxS motif and 47% of

the amino acids that code for the L3 loop (Fig. 2.3). Analyzing these embryos,

and Smad2ΔE9,10/+ and Smad23loxP/ΔE9,10 embryos, reveal a step-wise decrease in

the level of wild-type Smad2 mRNA transcript (Fig. 2.8); Smad23loxP/+ embryos

produced 69.2% wild-type mRNA, while Smad23loxP/3loxP produced only 39.2%.

With a reduction in mRNA, there is a correlated reduction in wild-type Smad2

protein [62]; thus, even though the hypomorphic allele produced less wild-type

Smad2 protein, there was enough to allow the embryo to develop and live.

23 However, producing less than 39.2% wild-type Smad2 mRNA was detrimental to

development; thus Smad23loxP/ΔE9,10 embryos, which produced only 27.7% (Class

III), were not viable after E10.5 (data not shown).

This new hypomorphic allele will prove to be extremely useful for determining novel functions of Smad2 in different developmental processes.

2.2.3 Conditional Removal of Smad2

In order to determine what function, if any, Smad2 has later in development, the Smad2flox conditional allele was used in conjunction with

various Cre mouse lines to recombine the Smad2 allele into the null allele. This

recombination will remove Smad2 in specific tissues and developmental stages,

depending upon the Cre line used. The Smad2flox allele was used instead of the

Smad23loxP conditional allele due to the hypomorphic nature of the allele and that

the combination of the Smad23loxP and Smad2ΔE9,10 alleles will produce a

phenotype with or without Cre recombinase present. One of the Cre transgenic mouse lines that was utilized was Mox2cre (also known as the MORE mouse

line), which expresses Cre recombinase at E5.0 in the epiblast. Thus, Cre would

be present only in derivatives of the primitive ectoderm by E7.0 [68]. This Cre

line does display slight mosaic activity, so some DNA is left unrecombined [68].

Mox2cre/+ was bred to Smad2ΔE9,10/+ to produce Smad2ΔE9,10/+; Mox2cre/+ animals.

The males were kept to breed with Smad2flox/flox females to analyze the effects of

Smad2 removal in embryonic, but not extra-embryonic tissues. In confirmation of

the importance of Smad2 in early development, no live-born Smad2flox/ΔE9,10;

Mox2cre/+ animals were ever identified (Table 2.4A). Analyzing embryos from 24 E8.5-10.5, Smad2flox/ΔE9,10; Mox2cre/+ embryos could be identified (Table 2.4B).

Unlike the three classes of disorders displayed by Smad23loxP/ΔE9,10 embryos,

Smad2flox/ΔE9,10; Mox2cre/+ embryos were grouped only into Class III because they

displayed midline defects and holoprosencephaly exclusively (data not shown).

The effects of Smad2 removal from endothelial tissue was also addressed using the Tie2-Cre transgenic mouse line. The Tie-2 promoter drives Cre

expression in a pan-endothelial-specific pattern, and Cre expression can be

observed in the endocardium and the endothelial cells of the aorta [69]. TGF-β

signaling is involved in vasculogensis, where the endothelial cell progenitors

differentiate and begin to form blood vessels [70], and in maintaining vascular

homeostasis [71]. In order to determine if Smad2 may be necessary for this

process, and to determine if Smad2 removal in endothelial cells would be lethal,

Smad2ΔE9,10/+; Tie2-Cre males were generated to breed with Smad2flox/flox females. Smad2flox/ΔE9,10; Tie2-Cre could be observed as liveborns (Table 2.4C) and had no apparent health problems upon weaning (postnatal day 28); embryos

(Table 2.4D) also had no apparent defect of any kind (data not shown).

Smad2flox/ΔE9,10; Tie2-Cre individuals appear to occur at less than Mendelian

frequency, but this may be due to small sample size.

Based on prior work done in the lab involving the conditional removal of

the common Smad, Smad4, in fibroblast and mesenchymal cells [72] using the

Fsp-Cre (A. Trimboli and G. Leone, unpublished data) mouse line, TGF-β

appears to be involved in hair follicle development and cycling (M. Hester, Ph.D

Dissertation). In addition, Smad4flox/flox; Fsp-Cre mice display small ears,

25 deformed digits, and reduced body size (M. Hester and M. Festing, unpublished

data) compared to their wild-type littermates. Since Smad2 and Smad4 coordinately regulate the expression of various genes involved in many aspects of development, it would be informative to know if conditional removal of Smad2 would result in a similar phenotype. Smad2flox/+; Fsp-Cre males were mated to

Smad2flox/flox females and Smad2flox/flox; Fsp-Cre animals were monitored for any

apparent lethality or physical abnormality. All the mice were viable (Table 2.4E),

healthy, and fertile, with none of the phenotypes observed in Smad4flox/flox; Fsp-

Cre mice.

2.3 Conclusions

As stated previously, deletion of the Smad2 N-terminus has been reported

to result in the production of a truncated protein [56]. In addition, a hypomorphic

allele of Smad2 has been reported, which phenocopies N-terminal Smad2 mutations [66]. Therefore, the previously published Smad2 conditional allele [58] could result in a hypomorphic allele after recombination. The Smad2 conditional alleles Smad23loxP and Smad2flox will be recombined in the presence of Cre into

the Smad2ΔE9,10 null allele. This allele is null due to the removal of part of the

MH2 domain which codes for the L3 loop and serine residues. This will provide a

powerful tool to analyze Smad2 function under a variety of physiological and pathological conditions. It has been reported that the neo cassette contains a cryptic splice donor and acceptor site and, therefore, insertion of a neo cassette intronically can result in a hypomorphic allele [66]. Even though homozygous

26 Smad23loxP mice are obtained at Mendelian ratios and show no observable

abnormality, the neo cassette is aberrantly spliced into the Smad2 mRNA

transcript. No functional protein is predicted to result from this fusion, so there

would be an overall reduction in functional wild-type Smad2 protein. This is

believed to be the underlying explanation as to why no Smad23loxP/ΔC or

Smad23loxP/ΔE9,10 animals could be found born alive, those embryos did not

possess enough functional Smad2 protein to meet the various threshold

requirements for correct and complete development. Analysis of the embryos

showed that all the embryos die before E11.5, and display three general classes

of phenotypes which is most likely due to modifiers in the strain background

finely modifying the level of Smad2 protein that is active. The fact that these

embryos did not all die of gastrulation defects, as observed in Smad2ΔE9,10/ΔE9,10 embryos indicates that an initial Smad2 threshold is present around gastrulation, and another exists before E10.5. Smad23loxP/ΔE9,10 embryos possessing 40% or

less wild-type Smad2 achieve the lower Smad2 threshold needed to pass

through gastrulation, but do not have enough for developing proper

anterior/posterior polarity or correctly patterning structures along the midline.

The idea of threshold, or relative dosage of the TGF-β Smads, has been

postulated before to explain the particular phenotypes observed [34, 62] (A.

Chow, honors thesis). This threshold concept is reinforced by observing the

phenotypes found in Smad2flox/ΔE9,10; Mox2cre/+ embryos, in which Smad2 gene

expression is removed around the time of gastrulation. There is enough Smad2

to develop through gastrulation, but not enough Smad2 available for the next

27 threshold, and thus, results in embryonic lethality before E10.5. These embryos

display the milder phenotypes that were observed in Smad23loxP/ΔE9,10. An explanation for this observation is that the Smad2flox/ΔE9,10; Mox2cre/+ embryo is a

mosaic, still retaining in enough individual cells sufficient amounts of Smad2 to

prevent developmental arrest in the earliest stages which would result in Class I

and Class II defects. To correct this, using the similar Sox2cre transgenic line

should completely remove Smad2 early in development and not leave the

embryonic portion mosaic [73]. Removal of Smad2 later in development, or in

adult fibroblasts, did not result in any observable phenotype. Assuming that the

recombination was efficient in these cells, it does not appear that Smad2 plays a

necessary role in these various tissues. Either Smad2 function is not needed at

these later stages of development, Smad2 had not been completely removed, or

there is another gene compensating for Smad2 loss, possibly the highly-related

Smad3.

2.4 Materials

2.4.1 Generation of Targeted ES cells and Creation of Germline Chimeric Mice

Previous work done by the graduate student Ye Liu resulted in a targeting vector containing a 9 kb Smad2 genomic fragment consisting of exons 6 – 11 subcloned into pBluescript KS (Stratagene, La Jolla, CA). A loxP site with an upstream EcoRV restriction site was inserted into the SalI restriction site in intron

8. A 3.1 kb EcoRI/EcoRI fragment was placed downstream of a floxed neo

cassette of pLoxpneo. A 5.9 kb EcoRI/NotI fragment was inserted upstream of

28 the floxed neo cassette. The NotI site was from the polylinker region of

pBluescript KS. The final result was to flank the genomic sequence of exons 9

and 10, as well as the neo cassette with loxP sites. This Smad2 3loxP vector was

electroporated into 129/SvEv TC1 ES cells [74] and selected with G418 and

gancyclovir. 192 resistant colonies were then selected and further analyzed by

Southern blot for correct targeting. Correctly targeted clones were microinjected

into C57BL/6 blastocysts, then implanted into foster mothers to obtain germline

chimeras by standard procedures. The chimeric founders were crossed with NIH

Black Swiss mice to obtain wild-type and Smad23loxP/+ offspring.

2.4.2 Southern Blot

Genomic DNA was extracted either from ES cell colonies or tail biopsy and digested with EcoRI and EcoRV (Invitrogen), which will produce an 11.4 kb wild-type fragment and an 8.5 kb targeted fragment. Digests were run overnight on 0.7% agarose gels containing less than 0.1% EtBr, then prepared for vacuum transfer to nitrocellulose filters (Hybond). The gel was soaked for 20 minutes in depurination solution (0.25M hydrochloric acid [HCl]), rinsed in d2H2O, then

soaked twice in denaturing solution (0.5M sodium hydroxide [NaOH] and 1.5M

sodium chloride [NaCl]) for 20 minutes each. The nitrocellulose membrane was pre-wet in d2H2O, then soaked for 5 minutes in denaturing solution. Genomic

DNA in the gel was then transferred to the nitrocellulose by vacuum transfer

using a Vacugene pump vacuum apparatus (Phamacia) for 1.5 hr. Nitrocellulose

was then soaked in 0.5M phosphate buffer (0.5M disodium hydrogen phosphate

29 [Na2HPO4] and 0.5M sodium phosphate monobasic [NaH2PO4•H2O]) for 3 minutes, wrapped in plastic wrap, UV cross-linked, baked at 80oC for 2 hours

between Whatman filter paper, and finally soaked in hybridization solution (0.5M

phosphate buffer, 7% sodium dodecyl sulfate [SDS], 1mM

ethylenedinitrilotetraacetic acid [EDTA], and 100 µg/ml denatured salmon sperm

DNA [Gibico]) for 3 hrs at 60oC. Radioactive probes were labeled using the

random primer labeling kit (Roche) and 32P-dATP, and hybridized 12-18 hrs in

hybridization solution at 60oC. The following day membranes were washed in

0.1M and 0.04M phosphate buffers with 0.1% SDS at 60oC. Membranes were

heat-sealed into a plastic bag and exposed to Biomax MR film (Kodak) overnight

at -80oC in a light-tight cassette. The film was then warmed slowly up to room temperature, and developed on a SRX-101A blot processor (Konica Minolta).

2.4.3 Mice and Matings

Mice were maintained on an outbred stock mix of 129/SvEv NIH Black

Swiss via interbreeding of related mice to produce live progeny of the following genotypes: wild-type, Smad23loxp/+, Smad23loxP/3loxP, Smad2flox/+, Smad2flox/flox,

Smad2flox/ΔE9,10, Smad2+/ΔE9,10, and Smad2flox/ΔC. These breedings were done by housing one male and no more than two females per breeding cage; these mice were given standard pellet food and water ad libitum. Genotyping of progeny was done by tail or toe biopsy in accordance with this lab’s approved animal protocol for acquiring tissue from live animals. To examine embryonic development, female mice at least 6 weeks of age were paired with a male of at

30 least 3 months of age and the females were checked daily for visualization of a

vaginal plug. The day of observation of the vaginal plug was considered

embryonic day 0.5. Sacrificing of research mice was done either by CO2 necrosis or cervical dislocation.

Once appropriate stage of embryonic development has been reached, the embryos were dissected from the maternal deciduas in 1x PBS pH 7.4, and extraembryonic membranes are removed. For genotyping, extraembryonic membranes or embryonic tissues are used. Gross evaluation of embryos were performed using a Zeiss Stemi SVII Apo dissecting microscope, and embryo pictures were taken using a MTI 3CCD digital camera attachment and Scion

Series 7 software. Embryos are then placed in a 4% PFA solution (1x PBS pH

7.4, 4% PFA, 0.5% Tween-20) to gently agitate at 4oC overnight. The following day the embryos were washed in 1x PBS, then dehydrated using an EtOH or

MeOH series (25%, 50%, 75%, 100%), washing in each concentration of alcohol for 1 hr at room temperature. Dehydrated embryos were then stored in 100% alcohol at -20oC. Whole deciduas were collected as mentioned, but embryos

were not removed. Deciduas were immediately fixed in 4% PFA and dehydrated

in an EtOH series at room temperature and stored at -20oC.

2.4.4 Genotyping

Genotyping of mice was performed by PCR on either tail or toe biopsy, or

Southern blot on tail biopsy, while genotyping of embryos was on extraembryonic

membranes, or embryonic tissue. For genotyping primers used, see Table 2.2.

31 The following primers were used to genotype these alleles: WT: SM2-16 and R2,

LEFT and SM2-AA; Smad23loxP: SM2-16 and R2, 3JNEO and SM2-AA;

Smad2flox: SM2-16 and R2, LEFT and SM2-AA; Smad2ΔE9,10: SM2-16 and WTR;

Smad2neo,ΔE9,10: SM2-16 and RINA; Cre: Cre-Y1 and Cre-Y2; Smad2ΔC: Fu-1 and

RINA. For PCR, standard reaction conditions were used in a total volume of

20µL using Taq polymerase (Invitrogen) and d2H2O in a PTC-1000 Peltier

Thermal Cycler (MJ Research). The reaction parameters are as follows: 5

minutes denaturation at 95oC, 35 cycles of 95oC for 30 seconds followed by the

primer specific annealing temperature of 60oC for 30 seconds followed by 72oC for 30 seconds, and a final 10 minutes at 72oC elongation step. PCR products

were visualized by running the fragments out on 1.5% agarose gels containing

less than 0.1% EtBr and visualized under UV light. Pictures were taken using

UVP ImageStore and printed out on a video copy processor P67U. For Southern

blot protocol, see section 2.4.2.

2.4.5 Histology

Whole deciduas were processed for sectioning by first going though two

100% EtOH washes, then two xylene washes for 20 minutes each. Deciduas

were then incubated for 1 hr in a solution of 1:1 xylene and paraffin at 57oC.

Finally, deciduas were incubated three times in paraffin at 57oC for 1 hr each.

After paraffin had perfused completely, deciduas were embedded into a paraffin

block that was then attached to a wooden block. The paraffin block was then

sectioned to a thickness of 5-8 µm using a Reichart HistoSTAT Rotary

microtome. The sections were then placed onto TESPA (3-

aminopropyltriethoxysilane [Sigma Chemical]) coated slides and dried for 1-2

days at 37oC.

2.4.6 Hematoxylin and Eosin Staining

Removal of paraffin and hydration of the slides was carried out by the following at room temperature: two 5 minute xylene washes, two 3 minute 100%

EtOH washes, one 3 minute 95% EtOH wash, one 3 minute 70% EtOH was, and

one 5 minute wash in dH2O. DNA in the nuclei was stained by Harris

Hematoxylin by dipping the slides for 15 seconds, dipping in dH2O, then rinsing under tap water for 5 minutes. Slides were then submerged in acidic 70% EtOH until a noticeable color change in the alcohol was detected, then neutralized in

basic 70% EtOH, then rinsed in tap water for 5 minutes. To visualize protein,

slides were dipped three times in 70% and 95% EtOH, stained in Eosin Yellowish

for 20 seconds, then rinsed by dipping 5 times into 95% EtOH. Slides were

further processed by two 5 minutes washed in 100% EtOH and two 5 minute

washes in xylene. Coverslips were then mounted using an organic solvent of 1:1

xylene and Permount and the slides were allowed to dry overnight in the

fumehood. Once dry, slides were evaluated using a Zeiss Axioskop2 light

microscope and photographed by a MTI 3CCD digital camera and Scion Series 7

software.

33 2.4.7 Xenopus laevis Injection Assay

Xenopus laevis eggs were fertilized in vitro and raised in 0.1x modified

Barth’s saline (MBS). Microinjections were performed in 1x MBS with 4% ficoll.

Embryos were injected into the subequatorial dorsal marginal zone at the 4-cell

stage with 1.5 ng of synthetic mRNA (produced by in vitro transcription using the

mMessage mMachine kit [Ambion, Austin, TX]) encoding either the dominant-

negative Smad23SÆA, hSmad2ΔE9-11, or hSmad2ΔCTD along with 0.5ng β-

galactosidase mRNA as a lineage tracer. The Smad23SÆA had the C-terminal phosphorylatable serines replaced by alanines, while the Smad2ΔE9-11 allele truncates after exon 8, thus removing a majority of the MH2 domain along with the phosphorylation sites. The hSmad2ΔCTD was constructed by truncating the

Smad2 wild-type transcript immediately before the phosphorylatable serines. All

Smad2 constructs contain exon 3. Over 100 Xenopus blastulae were injected

with the above mRNAs and phenotypically scored. To qualitatively measure the

differences observed in injected embryos, the dorso-anterior index (DAI) was

used [64], which allows one to assign a numerical score based on varying

degrees of dorsalization or ventralization. Lineage tracing with β-galactosidase

(stained with either Red-Gal [{6-Chloro-3-indolyl-β-D-galactoside} resulting in a

red precipitate] or X-Gal [{5-bromo-4-chloro-3-indoyl β-D-galactopyranoside}

resulting in a blue/teal precipitate]) confirmed the injection into dorsal areas of the

embryos. Western blots on neurula stage embryos were performed using

34 standard procedures and probed for the FLAG epitope attached to the N-

terminus of Smad2 using a M2 anti-FLAG antibody from Sigma. A nonspecific

band was used as a loading control.

2.4.8 RNA Extraction

Timed matings were set up as described in section 2.4.3. To acquire

Smad23loxP/3loxP , Smad23loxP/+, and wild-type embryos, Smad23loxP/+ males and females were paired together. Smad2ΔE9,10/+, and Smad23loxP/ΔE9,10 siblings were

generated through mating a Smad2ΔE9,10/+ male with Smad23loxP/+ females.

Smad2ΔC/ΔC samples were isolated from tissue culture cells, since Smad2ΔC/ΔC embryos are too small to acquire useful amounts of RNA for analysis.

Dissections were performed in the mornings to acquire E10.5 embryos, which were placed into individual tubes and the corresponding extraembryonic portions were retained for genotyping. Embryos were flash-frozen on metal cooled to

-150oC via liquid nitrogen. Once genotyping was complete, littermates of the same genotype were pooled to increase yield for RNA extraction. Tissue was homogenized in 500 µl of TRIzol reagent (Life Technologies) by repeated pipetting of the samples through an RNAse-free pipette tip, and incubated at room temperature for 5 minutes. 100 µl of chloroform was added and shaken vigorously by hand for 15 seconds, then incubated at room temperature for 3 minutes. The phases were separated by centrifugation at 12, 000 x g for 15 minutes at 4oC. Aqueous phase was isolated into a new tube and 250 µl of isopropanol was added, mixed, incubated at -80oC for 10 minutes, then

35 centrifuged at 12, 000 x g for 10 minutes at 4oC. Supernatant was poured off

and the RNA pellet was washed in 70% EtOH in DepC water, then centrifuged at

7, 500 x g for 5 minutes at 4oC. EtOH was poured off and the RNA pellet allowed

to air dry, then resuspended in 20 µl of DepC water. To ensure no genomic DNA

contamination, samples underwent DNaseI treatment by adding together 1 µg of

RNA sample, 1x DNase reaction buffer, 10 units of DNaseI, all diluted in DepC water. This was incubated at room temperature for 15 minutes, then 1 µl of 25 mM EDTA (ethylenediaminetetraacetic acid) solution was added and incubated at 65oC for 10 minutes.

Up to 5 µg of DNaseI-treated RNA was used to make cDNA using the

First-strand cDNA synthesis kit (Invitrogen) following manufacture’s procedure and using oligo(dT)20 primers provided in the kit. 500 ng of synthesized cDNA was used in all RT-PCR reactions. PCR products were visualized by running the fragments out on 1.5% agarose gels containing less than 0.1% EtBr and visualized under UV light. Pictures were taken using UVP ImageStore and printed out on a video copy processor P67U. See Table 2.2 for primers used.

The following primers sets were used: WT: E8L and CTNeo2; Smad2-neo: E8L

and WTR; HPRT: HPRT L and HPRT R.

2.4.9 Real-Time PCR

Real-time PCR was done on an iCyclerH iQ Real-Time Detection System

using iQTM SYBR® Green Supermix (Bio-Rad) available from the Plant-Microbe

Genomics Facility located at 420 Biological Sciences Building. D2 water and

36 RNA used to make the cDNAs were included as controls. All samples were repeated in duplicate and normalized against the loading control L34. Samples that did not have more than 10 cycles difference between cDNA and RNA cycle number were removed from the analysis. Wild-type was considered 100% and all samples are compared as relative to wild-type levels. For primers used, see

Table 2.2. The following primers sets were used: WT: Sm2E10.5F and pR; L34:

L34 1 and L34 2.

Table 2.1 Heterozygote interbreeding of the Smad2 alleles. Smad23loxp/3loxP and Smad2flox/flox were able to be produced at Mendelian ratios at postnatal day (P) 21. Smad2ΔE9,10/ΔE9,10 mice are not viable.

Table 2.2 Primer sequences.

Table 2.3. Intercrosses of the conditional alleles to the null allele. Genotypic analysis of offspring resulting from crosses between: A. Smad23loxP/3loxP and Smad2ΔC/+, B. Smad23loxP/3loxP and Smad2ΔE9,10/+, C. Smad23loxP/3loxP and Smad2ΔE9,10/+, and D. Smad2flox/flox and Smad2ΔE9,10/+. A, B, and D are summaries of live born offspring, while C summarizes embryo offspring.

Table 2.4. Intercrosses of Smad2flox/flox to various Cre mouse lines. Genotypic analysis of: A. live born using Mox2cre, B. embryos using Mox2cre, C. live born using Tie2-Cre, D. embryos using Tie2-Cre, and E. live born using Fsp-Cre.

Figure 2.1 Generation of the Smad23loxP allele and mouse. A. Genomic sequence containing exons 6–11 was subcloned for vector construction. The Smad2genomic locus is shown to scale at top while, the targeting vector is shown in the middle, and the targeted allele (3loxP) is at the bottom. Exons 6–11 are indicated by solid boxes. B. Homologous recombinants were identified by Southern blot analysis. Genomic DNA from ES cell clones were digested with EcoRI and EcoRV and hybridized with a 5’ external probe, which detects a wild- type 11.4 kb fragment and a targeted 8.5 kb fragment. C. Genomic DNA from mice was the same as in B. Smad23loxP/+ mice have both these fragments, while Smad23loxP/3loxP mice have only the targeted 8.5 kb fragment. Adapted from [75].

Figure 2.2 Alleles from Cre-mediated recombination of Smad23loxp and the PCR strategy used to identify them. A. Founder mice heterozygous for the 3loxP allele were bred with EIIa-Cre mice to obtain offspring, which were mosaic for the different recombination alleles. Red arrowheads indicate the location and direction of the primer sets used for identification of specific alleles. The Smad2 genomic loci are shown to scale at the bottom; regions of the protein corresponding to the different exons are indicated by dashed lines at the top. Adapted from [75].

Figure 2.3 Protein sequence of various Smad2 products. Smad2 wild-type protein sequence is on top; green line indicates the L3 loop and the red line indicates the phosphorylation site. The middle sequence corresponds to the predicted protein sequence from the Smad2ΔE9,10 null allele, if translated. Due to the removal of exons 9 and 10, exon 11 is out of the reading frame and truncates early. The bottom sequence corresponds to the predicted protein sequence from the Smad2-neo mRNA, if translated. Insertion of the neo cassette into the mRNA results in an early stop codon because neo is out of the reading frame. Note that only about half of the necessary residues are present that would form the L3 loop; thus the loop is incomplete and most likely nonfunctional. Residues coded by separate exons are alternatively black or grey, while red indicates residues encoded by two exons.

Figure 2.4 Analysis of Smad2ΔE9,10 allele. A. Embryos from Smad2ΔE9,10/+ intercrosses were observed at E7.5. Smad2ΔE9,10/ΔE9,10 embryos (iii) are much smaller than their wild-type (WT) littermate (i). The middle embryo (ii) is Smad2ΔE9,10/+, which has embryonic tissue external to the yolk sac. This defect is observed in less than 10% of the heterozygous. B. Section of embryos from Smad2ΔE9,10/+ intercrosses were observed at E6.5. Smad2ΔE9,10/ΔE9,10 embryos (right) are again much smaller than their wild-type littermate (left) and lack discernable structures. Adapted from [75].

Figure 2.5 Analysis of the Smad2ΔE9,10 allele compared to dominant-negative Smad2 constructs. Xenopus injections were used to compare the function of Smad2 mutant alleles. A. Uninjected control. B-C. Injection of the dominant- negative Smad23SÆA. D-F. Injection of hSmad2ΔE9-11. G-I. Injection of a mild dominant-negative hSmad2ΔCTD. J. Western blot of uninjected and injected Smad23SÆA (3SÆA) and hSmad2ΔE9-11 (ΔE9-11) Xenopus embryos showing the presence of the Smad2: flag fusion proteins. The lower panel shows the loading control. Only the hSmad2ΔE9-11 injected embryos displayed a normal phenotype, indicating this construct does not have antimorphic effects. Adapted from [75].

Figure 2.6 Smad23loxP/ΔE9,10 embryos are early embryonic lethal. Smad23loxP/ΔE9,10 E10.5 embryos display a range of phenotypes. A. Class I. B. Class II. C. Class III. D. Normal E10.5 Smad23loxP/+ littermate. Adapted from [75].

Figure 2.7 Aberrant splicing of the Smad23loxP allele. A. Graphical representation of wild-type mRNA and the alternative splicing which incorporates the neo cassette (red box) that is produced from the Smad23loxP allele. Alternating exons are blue and green boxes and exons 7-11 are labeled. B. Non-quantitative RT- PCR (left) of the Smad2-neo transcript (top), wild-type transcript (middle), and the loading control HPRT (bottom). To the right are graphical representations of the two Smad2 transcripts and primer and primer placement used to detect them. Only in E10.5 embryo samples which genotyped as having at least one Smad23loxP allele could the Smad2-neo transcript be detected.

Figure 2.8 Real-time PCR of wild-type Smad2 transcript levels found in various Smad2 genotypes. Wild-type is normalized to 100% and all samples are considered percentage relative to wild-type. Smad23loxP/3loxP produced 69.6%, Smad2ΔE9,10/+ produced 50%, Smad23loxP/3loxP produced 39.2%, Smad23loxP/ΔE9,10 (Class III) produce 27.7%, and Smad2ΔC/ΔC produced no detectable wild-type Smad2 transcript. Sm is an abbreviation for Smad.

CHAPTER 3

REMOVAL OF SMAD2 RESULTS IN INCREASED SENSITIVITY TO

RETINOIC ACID INDUCED HOLOPROSENCEPHALY

3.1 Introduction

Retinoic acid, the biologically active metabolite derivative of Vitamin

A/retinol, is important for many developmental processes. Like many vitamins, too much or too little of vitamin A has detrimental effects upon development [76].

Vitamin A, in particular, can easily penetrate into the developing CNS causing irreparable harm [77]. Thus, throughout development, the levels of Vitamin A are rigorously maintained, with the liver functioning as a reservoir [78]. At weaning

(around three weeks of age) a mouse has already stored enough Vitamin A in its liver to last a lifetime [78].

Retinoic acid is vital to the normal development of the cranial neural crest, heart, respiratory tract, limb formation, skeletal development, specification of the anterior-posterior axis, and patterning of the CNS (reviewed in [79]). Retinoic acid induces the formation of neurons and glia, and can increase the number and

50 length of neurites [80]. During development, all-trans retinoic acid (atRA) is

almost absent from the forebrain, midbrain, and hindbrain. The spinal cord of the

embryo contains the most atRA [81]. Thus, it has been proposed that the atRA

present in the spinal cord diffuses into the caudal hindbrain, where gene

transcription and development is altered. To keep the atRA from diffusing further

into the rostral brain and exposing cells inappropriately to RA, it is catabolized

[82]. Fetal exposure to excess RA in humans via topical exposure to Retin-A,

Vesanoid, and tretinoin on the mother, resulted in abnormalities of the CNS [83,

84, 85].

3.1.1 Retinoic Acid Metabolism

Individual cells are capable of creating and regulating the level of retinoic acid that are available to the cell and the surrounding area, and does so beginning at E7.5 in the mouse [86]. Synthesis into RA from retinol is a two-step process involving two enzyme families that have recently specialized in metabolizing retinol in chordates, possibly emerging at the same time as the dorsal neural tube [87] (reviewed in [88]). Retinoic acid metabolism can be reviewed in [89, 90, 91, 92] and Fig.3.1.

In mammals, retinol is oxidized into retinaldehyde by alcohol dehydrogenase 4 (ADH4), while retinaldehyde is metabolized into atRA by aldehyde dehydrogenase 1, 2, and 3 (RALDH1-3); the other thirteen aldehyde dehydrogenases are not able to catalyze this reaction. The conversion of retinol to retinaldehyde is reversible and the rate-limiting step [93], while the conversion

51 of retinaldehyde to retinoic acid is not. All mammals possess 4 types of alcohol

dehydrogenases (ADH1-4), though only ADH1 and ADH4 have been

demonstrated to be capable of oxidizing retinol in vitro, and ADH4 is 10-100 fold

more efficient than ADH1 [94, 95]. Binding of retinol to ADH4 can be

competitively inhibited with high doses of ethanol in vitro [96] and results in

reduction of retinoic acid in vivo [97].

Retinoic acid can be degraded into 4-hydroxy-retinoic acid and 4-oxo- retinoic acid by use of the cytochrome P450 proteins, a diverse group that are responsible for the metabolism of both exogenous (such as xenobiotics) and endogenous products (such as steroids) [98, 99]. Specifically, cytochromes

P450RA-1 (Cyp26A1), P450RA-2 (Cyp26B1), and Cyp26C1, are important in the embryo for regulating the quantity of retinoic acid.

All these enzymes can be found in various stages of embryonic development. When components are removed from the mouse, embryonic defects and lethality occur. Removal of RALDH2 or Cyp26A1 is lethal [100, 101]; however, removal of ADH4 is not lethal, but the embryos are very sensitive to fluctuations in RA level [102]. It may be possible that ADH4-/- embryos are able

to survive due to compensation from ADH1.

3.1.2 Retinoid Signaling

Retinoid signaling utilizes two families of nuclear ligand-activated

transcriptional regulators, the retinoic acid receptor (RAR) and the retinoid X

receptor (RXR), (reviewed in [79, 103]) (Fig. 3.2). The RARs and RXRs each

52 have three family members (α, β, and γ), all encoded by separate genes, and

splice variants of these family members which arise due to differential use of two

promoters found at most of these genes. The RARs and RXRs are either

heterodimers or homodimers that are bound to target sequences in the genome

via a DNA binding domain in the protein, and the RARs and RXRs interact with

HDACs. The core DNA binding motif consists of direct repeats of

PuG(G/T)TCA(X)nPuG(G/T)TCA, or a slight variation, with n referring to the number of nucleotides between the repeats. Nucleotides of 2 or 5 (DR-2 and

DR-5 respectively) are considered RAREs that RARs bind to, while only 1 nucleotide between the repeats (DR-1) is an RXRE that RXRs bind to. The

RARs are active when bound to atRA and 9cis-RA, while RXRs are active when bound to 9cis-RA. Once RA has been bound to, the RARs and RXRs undergo a conformational change, which allows association with HATs, and gene transcription takes place due to the chromatin decondensation. A reversed orientation of RAR:RXR heterodimer on the DNA can result in constitutive gene repression. All RARs and RXRs are expressed in various tissues throughout development. This variety, and variety in possible heterodimer combinations, allow for specificity of gene regulation (reviewed in [79, 103]).

There are over 500 known targets of retinoid signaling that have been observed to be enhanced, repressed, or both, in the presence of RA [104].

Additionally, retinoid signaling regulates the expression of the RARs and RXRs, and the enzymes involved in RA metabolism and degradation. This creates a feedback loop that allows the cell to regulate and maintain the amount of

53 biologically active RA and retinoid signaling levels. Removal of individual RAR

(α, β, or γ) and RXR (α, β, or γ) genes via gene knockout result in mild phenotypes, while compound deletions of RARs, RXRs, or RAR/RXRs (α, β, or

γ) result in Vitamin A deficiency (VAD) syndrome defects [105, 106].

3.1.3 Retinoid Signaling Affects TGF-β Signaling

Both TGF-β and retinoid signaling are vital to the correct development and viability of embryos. Researchers have observed that retinoid signaling can regulate TGF-β signaling [107, 108, 109, 110, 111]. Exposure of mouse embryonic palate mesenchyme to retinoic acid resulted in an increase of TGF-β3 mRNA, a decrease in TGF-β type II receptor mRNA, a decrease in activated

Smad2 and Smad3, and an increase in Smad7 protein [107]. Exposure of atRA also resulted in a reduction of active Smad2 and Smad3, an affect dependant on

RARα [108]. Treatment of mouse inner ear mesenchyme with retinoic acid resulted in a decrease of TGF-β1 ligand [109], and a decrease in TGF-β type II receptor and Smad2 [110]. Additionally, using a mouse line with a targeted deletion of TGF-β2 ligand, a relationship was observed between the genotype of the embryo and the dam as an indicator of atRA-induced cleft palate [111].

When the dam was wild-type, 48% of the wild-type embryos displayed cleft- palate, compared to 71% of the heterozygous littermates. When the dam was a heterozygote, the wild-type and heterozygous embryos displayed cleft-palate at

54 nearly the same penetrance (74 and 77%, respectively) [111]. Thus, TGF-β signaling can be influenced by RA and retinoid signaling, and removal of a TGF-β

signaling component results in sensitivity to atRA teratogenesis.

3.2 Results

3.2.1 Retinoic Acid Signaling is Affected in Smad2 Embryos

Since TGF-β signaling can be influenced and regulated by RA and

retinoid signaling, it was essential to determine if retinoid signaling could be

influenced and regulated by TGF-β signaling. Since Smad2 is important for

embryonic development [56, 62, 75], analysis was performed to determine if a

reduction of Smad2 had any effect upon retinoid signaling. Retinoid signaling

level and domain can be visually assayed using a reporter mouse line (TGII) that

has an artificial promoter consisting of three tandem RAREs (amplified from the

RARβ gene, one in the forward orientation, two are reversed) upstream of a LacZ

cassette (Fig. 3.3A), which was randomly inserted into the mouse genome [112].

Staining intensity for β-galactosidase correlates with the retinoid signaling

domain and retinoid signaling level; the strain is also responsive to increases in

retinoid signaling induced by atRA [112].

This reporter mouse line was bred to Smad2ΔE9,10/+ mice to visualize

retinoid signaling levels and domain. Combining data from all litters, it was

observed that at E9.5, 87% of wild-type embryos had average staining intensity,

while the remaining 13% were darker (n=24) (Fig. 3.3B and D). The variation

observed in wild-type embryos is most likely due to unknown modifiers in the

55 mixed background that the mice were maintained on, or natural self-regulation

that was occurring at the time of fixation. Smad2ΔE9,10/+ littermates, however,

displayed greater variation in staining intensity with 8% staining noticeably lighter

than average and 40% had darker staining (n=25). Thus, at E9.5, retinoid

signaling is already becoming misregulated and misbalanced. By E10.5, all the

wild-type embryos were observed to have the staining intensity of average (n=4),

only half of the Smad2ΔE9,10/+ embryos displayed similar average staining intensity to wild-type. The remaining embryos were evenly split between higher

or lower staining intensity (n=8) (Fig. 3.3C and E). Thus, when wild-type

embryos are all able to normally self-regulate their retinoid signaling, only half of

the Smad2ΔE9,10/+ embryos are able to maintain average retinoid signaling levels.

The general domain of staining was similar, indicating that retinoid signaling was

not occurring in ectopic locations, just fluctuating in endogenous locations.

When β-galactosidase expression level was quantified and normalized against total protein, wild-type E9.5 embryos averaged 1.02 ± 0.61, while

Smad2ΔE9,10/+ embryos from the same litters averaged 0.79 ± 0.87. Though there

is overlap between these two values, what is observed is that the deviation of

these values is greater in the heterozygous embryos than in the wild-type embryos. Thus, quantifying β-galactosidase levels from embryos at E9.5 indicate that β-galactosidase, and hence retinoid signaling levels, are varying greatly when compared to variation found in the wild-type littermates. Alternatively, this

may be the result of small sample size, as there is a total n<8.

56 3.2.2 Sensitivity of Smad2 Embryos to Retinoic Acid Teratogenesis

It has been previously reported that reductions in TGF-β signaling

components resulted in embryos sensitive to RA teratogenesis [111]. In order to

determine if reduction in Smad2 also resulted in sensitized embryos, a RA

teratogenic assay was performed. Wild-type and Smad2ΔE9,10 heterozygous

embryos were exposed in utero to atRA at a dose of 7.5 mg/kg of maternal weight by intraperitoneal (IP) injection at E7.5 [113]. Previously, this teratogenic assay revealed that 70% of Smad2ΔC/+ embryos displayed HPE, compared to none of the wild-type siblings (T. Carrel, Ph.D. Dissertation). When this same assay was repeated later, Smad2ΔE9,10/+ embryos still displayed sensitivity to high

levels of exogenous atRA, but the percentage affected with HPE dropped to

25%, while HPE in wild-type embryos increased to 5% (Fig. 3.4A). Several possible explanations for the discrepancy of embryos affected with HPE between the two assays can be suggested to include: variation in injection technique, variation due to different batches of atRA and DMSO, differences between the weight and health of the mice involved in the teratogenic assay, differences in the animal facility that may have contributed an unknown variable that affected the

HPE penetrance, differences in the uncharacterized modifiers present the strain backgrounds, or use of different Smad2 null alleles. Different batches or animal facility environment were variables that could not be changed or reconstructed.

The general health of the mice are monitored rigorously, so should be similar over the years. Injection technique can vary from person to person, and all attempts were made to have the original person performing the IP injection

57 instruct the next individual, who was solely responsible for all subsequent

injections. The two Smad2 null alleles have the same affect on development,

and can be considered interchangable (Chapter 2). Differences in the strain

background could explain the discrepancy, as the mouse strain in the colony changes over the years. Even though there were differences in the actual percentage of HPE embryos observed, Smad2ΔE9,10/+ embryos still were more

sensitive to atRA teratogenesis than their wild-type siblings.

This increased sensitivity can also be observed when utilizing the

hypomorphic Smad2 allele. It was hypothesized that if Smad2 is affecting the

embryos ability to survive exposure to increased levels of atRA, then modulating

the amount of wild-type Smad2 should have a corresponding affect on the

penetrance of HPE in these embryos. We observed that embryos harboring a

single copy of the hypomorphic allele Smad23loxP had about 30% penetrance of

HPE after exposure to atRA (Fig. 3.4B). Hypomorphic homozygotes, which

would have less Smad2, were more sensitive to the exogenous atRA, and nearly

80% of the embryos displayed HPE. No direct correlation between the

penetrance of HPE in the Smad2ΔE9,10/+ embryos and the penetrance of HPE in

the Smad23loxP/+ can be determined. Based on real-time PCR performed (Fig.

2.8), Smad2ΔE9,10/+ should have less Smad2 than Smad23loxP/+ embryos, and as

such, should be more sensitive to RA-induced HPE if there is a direct correlation

between Smad2 levels and sensitivity, as is suggested when comparing

Smad23loxP/+ embryos to Smad23loxP/3loxP embryos. The RA teratogenesis

summarized in Figure 3.4 A and B was performed on different strain

58 backgrounds, as the mice used were from separate sub-populations of mice within the colony. Additionally, there are no wild-type sibling embryos to compare to Smad23loxP/+ embryos, which would have provided a baseline comparison of change of HPE penetrance relative to wild-type, which was done with the Smad2ΔE9,10/+ embryos. Since this was not how the experiment was designed, no conclusion of whether Smad23loxP/+ embryos are more or less sensitive to RA-induced HPE compared to Smad2ΔE9,10/+ embryos can be stated.

Based on the results of the separate RA teratogeneic assays, Smad2 is involved in determining how sensitive early-stage embryos will be to the teratogenic challenge of atRA. This sensitivity can be modulated by fluctuating the amount of wild-type Smad2 mRNA that will be produced, as can be observed when comparing hypomorphic heterozygotes to hymomorphic homozygotes.

Since Smad2 is involved in regulating gene expression, it is possible that

Smad2’s effect upon a downstream target gene(s) is a factor in the sensitivity to teratogenesis via atRA exhibited by the Smad2 embryos.

3.2.3 Retinoic Acid Metabolizing Enzymes ADH4 and Cyp26A1 are Affected in Smad2 Embryos

Based on observations that reduction in Smad2 results in increased sensitivity of embryos to exogenous atRA, and that retinoid signaling appears to be misregulated, a direct genetic role for Smad2 in retinoid signaling was investigated. Previously, observations suggested that Smad3 bound to RARγ in

59 vitro [114]. Smad2, being highly homologous to Smad3, was tested for any ability to also bind RARγ in vitro. No evidence of any interaction was identified

(S. Lasse, honors thesis).

Since Smad2 functions in regulating gene transcription, the transcription level of genes involved in retinoid signaling were investigated using a semi- quantitative RT-PCR approach at multiple stages of development before the forebrain had completely divided. Not all RARs and RXRs are expressed at every embryonic stage, but when they are, there was no detectable difference in expression level of these factors between Smad2 heterozygotes and wild-type siblings (Fig. 3.5). However, the sensitivity of Smad2 embryos to atRA and the misregulation of retinoid signaling indicated that this signaling pathway was somehow being affected. Since there was no apparent change in the receptors, attention turned to the metabolism of retinoic acid itself. Using the same samples as before, semi-quantitative RT-PCR was performed on various enzymes involved in this pathway. At most stages of development there was no detectable change in most of the genes analyzed (Fig. 3.6 and data not shown).

Two transcripts, however, were affected; Cyp26A1 was observed to only be reduced in the Smad2 heterozygotes at E8.5, while ADH4 was affected in every embryonic stage analyzed and always inverse to wild-type levels. Cyp26A1 is only reduced in E8.5, by E9.0, the gene transcription level is similar between wild-type and Smad2 heterozygotes. Adh4 gene expression is transitory and begins to be restricted at E9.5 in wild-type embryos [115, 116], which may explain why we do not detect ADH4 in our wild-type samples.

60 The affect on these gene transcripts was only observable in Smad2 heterozygous embryos, and was not indicative of general effects of TGF-β responsive Smads. Smad3 is highly related to Smad2, sharing over 92% homology due to these Smads arising by a gene duplication event unique to the chordate phyla [117]. However, these gene transcripts are not affected in

Smad3+/- embryos near-congenic on C57BL/6. It will still need to be determined if removal of all Smad3 transcript also has no effect on the transcription level of these genes, which would suggest that Smad2 is the only TGF-β R-Smad that can affect transcription of these genes.

Despite the observation that there appears to be no ADH4 transcript in

Smad2 heterozygous embryos at E8.5, there is in fact a very low level. This level simply may be too low to be visualized. Using real-time PCR on E8.5 embryos, there appears to be an almost 70% reduction in ADH4 compared to wild-type siblings (Fig. 3.7). The decrease of ADH4 in embryos reduced in Smad2 can also be confirmed using the hypomorphic allele. ADH4 transcript levels are reduced by 36% in Smad23loxP/3loxP embryos compared to Smad23loxP/+ siblings.

Since ADH4 and Cyp26A1 appear affected in Smad2 heterozygotes, it was possible that TGF-β signaling, through Smad2, could have a direct affect on regulating these genes. In order to determine this, the promoters of these two genes were scrutinized for the presence of tandem repeats of the SBEs. The

ADH4 promoter (Fig. 3.8) has two potential regions where a Smad could bind, one beginning at -1303 nucleotides upstream of the transcription start site and the other beginning at -6489. Multiple potential sites for retinoid signaling

61 regulation can be observed, both for transcription (DR5) and repression (DR1).

When the promoter of Cyp26A1 was analyzed (Fig. 3.9), multiple potential

tandem SBEs were observed between -3567 and -3997, and one at -2458

upstream of the start site. One potential site of retinoid regulation was also observed, a DR5 site. Thus, it is possible that these genes could be directly regulated by TGF-β.

3.3 Conclusions

It has been previously reported that reductions in TGF-β signaling components resulted in embryos sensitive to RA teratogenesis [111]. Reduction of Smad2 resulted in embryos that were more sensitive to RA-induced HPE.

This can be observed with both the null alleles and the hypomorphic alleles.

Using the TGII mouse reporter line, Smad2 heterozygous embryos display both increases and decreases in retinoid signaling levels. This is more extreme than what can be observed in wild-type littermates and indicates that misregulation of

retinoid signaling is taking place. This is in contrast to when the TGF-β and retinoid signaling corepressor Tgif is removed, as retinoid signaling only increased, never decreased (section 5.2.2). Since retinoid signaling appeared so misregulated, analysis was undertaken to determine if there were genes involved in retinoid signaling that was affected when Smad2 was reduced that could result in embryos sensitive to teratogenic levels of atRA. Two genes involved in the synthesis and degradation of retinoic acid, ADH4 and Cyp26A1, were found to be affected. The reduction of these gene transcripts early in development does not

62 result in lethality of the Smad2 embryos, as nearly all are born. However, these

embryos are sensitive to increases in RA. This sensitivity to fluctuations was

also observed in ADH4-/- embryos. They were born, but exposure to RA, or removal of vitamin A from the diet, resulted in embryos with abnormalities [102].

Thus these embryos could still survive, but when challenged, their ability to

adjust and survive is compromised. Thus it may be that the Smad2 embryos are

sensitive to RA because their ability to adjust quickly and correctly to RA

challenge is deficient, with the immediate underlying cause being the severe

reduction of ADH4.

Analyzing the promoters of the ADH4 and Cyp26A1 genes reveal

potential SBEs where TGF-β signaling could directly regulate these genes.

Considering Smad3+/- embryos did not show an effect in the transcription level of any gene analyzed, Smad2 is most likely to be mediating this regulation.

Differential use of available corepressors and coactivators may explain why

ADH4 expression can be both reduced and enhanced in Smad2 heterozygous

embryos, depending on what stage is being analyzed. This would be the first

reported instance of TGF-β signaling directly regulating the expression of genes

critical to retinoid signaling. Alternately, the presence of tandem repeats of SBEs

in the promoters of these genes may be coincidental, and Smad2 is regulating an

intermediate, which in turn is directly regulating these genes. Identifying tandem repeats of SBEs is rare, as none were observed in the promoters of the RARs,

RXRs, RALDHs, and other less prominent enzymes involved in retinoid signaling and metabolism (data not shown). Thus, the tandem SBE repeats observed in

63 the promoters of the ADH4 and Cyp26A1 genes are sites where TGF-β signaling can specifically regulate retinoid metabolism, and thus, regulate retinoid signaling.

3.4 Materials

3.4.1 Mice and Matings

Wild-type, Smad23loxP/3loxP, Smad23loxP/+, Smad2ΔC/+, TGII and

Smad2ΔE9,10/+ mice were maintained on a mixed background including 129SvEv,

NIH Black Swiss, and C57BL/6, or near-congenic to the F5+3 generation on

C57BL/6, if specifically stated. See section 2.4.3 for specific procedure for

matings and acquiring embryos for analysis. Embryos were stored in 100%

MeOH at -20oC until use. Embryos used for semi-quantitative or real-time RT-

PCR were handled as described in section 3.4.6.

3.4.2 Genotyping

Genotyping of mice was performed by PCR on either tail or toe biopsy,

while genotyping of embryos was on extraembryonic membranes, or embryonic

tissue. For genotyping primers used, see Table 2.2. The following primers sets were used: WT: SM2-16 and R2, LEFT and SM2-AA; Smad23loxP: SM2-16 and

R2, 3JNEO and SM2-AA; Smad2ΔE9,10: SM2-16 and WTR; Smad2ΔC: Fu-1 and

RINA. For alleles specific to this chapter, see Table 3.1. The following primer

sets were used: WT: Sm3-7 and Sm3-5; Smad3-: Sm3-5 and RINA (from Table

2.2); TGII: TG F and TG R. For PCR procedure, see section 2.4.4.

64 3.4.3 Teratogenic Application of Retinoic Acid

Timed mating was set up as described in section 2.4.3. To acquire

Smad23loxP/3loxP embryos, Smad23loxP/3loxP males and females were paired

together, while Smad23loxP/+ embryos were generated through matings of wild-

type males with Smad23loxP/3loxP females. Smad2ΔC/+, Smad2ΔE9,10/+, and wild-

type siblings were generated predominately through mating of the transgenic

male with wild-type females who were all of similar age and weight. On E7.5,

pregnant females were injected intraperitoneally with 5.0-7.5 mg of 1-2 mg/ml

retinoic acid in DMSO per kg of maternal body weight, as recommended in [113].

The atRA was also administered suspended in a 90/10 mixture of peanut oil

/DMSO and injected; DMSO and the atRA were suspended into the peanut oil by

vortexing immediately before injection. Embryos used for physical analysis or

sectioning were isolated at E9.5-10.5, and photographed and processed as

needed. Embryos to be used in RNA extraction were isolated between E8.5-9.5

and processed as described in section 3.4.9.

3.4.4 Whole Mount Visualization of β-galactosidase Expression

Timed matings were set up as described in section 2.4.3. Smad2ΔE9,10/+ and wild-type siblings were generated predominately through mating of the transgenic male with wild-type females. After dissection in 1x PBS, embryos were fixed in 4% paraformaldehyde with 0.2% glutaraldehyde for 5 minutes for

E8.5 embryos, and 15 minutes for E9.5-10.5 embryos. Embryos were then washed twice in 1x PBS. They were then incubated with mild shaking for 60

65 minutes at room temperature in the dark in β-galactosidase staining media (5mM potassium ferricyanide, 5mM potassium ferrocyanide, 1.0mg/ml X-gal [diluted in

DMF], 0.1% Tween-20, 2mM MgCl2, diluted in 1x PBS). Finally, embryos were

washed twice with 1x PBS and photographed by a MTI 3CCD digital camera and

Scion Series 7 software for visual comparison by only one person. Average

staining was determined by the staining intensity of the majority of the wild-type

embryos from each litter, to account for staining variation between solution

batches and between litters. Average staining intensity can be different between

litters. Once average staining level had been determined for a particular litter, all

embryos in that litter were compared and scored as either similar (average),

darker, or lighter.

3.4.5 Quantification of β-galactosidase expression

Timed matings were set up as described in section 2.4.3. Smad2ΔE9,10/+ and wild-type siblings were generated predominately through mating of the transgenic male with wild-type females. After dissection, embryo heads were removed just above the auditory vesicle and placed in 40µl of 1x Promega

Reporter Lysis Buffer (Promega). After homogenization via vortexing and repeat pipetting of the sample, 20μl was removed and added to 400µl of Z buffer (60mM

Na2HPO4•7H2O, 40mM NaH2PO4•H2O, 10mM KCl, 1mM MgSO4, 0.27% β-

mercaptoethanol, 4.3mM ONPG [Ortho-nitrophenyl-β-D-galactopyranoside] pH

7.0). This solution was incubated at 37oC for 6 hours. The reaction was

terminated by adding 200μl of 1M Na2CO3, and expression levels were

66 calculated with a spectrophotometer by measuring the absorbancy at 420 nm.

These values were normalized against the total protein concentration of each

D sample, which was measured using the BioRad C Protein Assay Kit (BioRad)

according to manufacturer’s protocol and absorbancy was measured at 750 nm.

3.4.6 RNA Extraction and Analysis

Timed mating were set up as described in section 2.4.3. Smad2ΔC/+,

Smad2ΔE9,10/+, and wild-type siblings were generated predominately through mating of the transgenic male with wild-type females. Smad3+/- and wild-type

+/- siblings were generated by mating a wild-type F5+3 mouse to a Smad3 F5+3 mouse. Dissections were performed in the mornings to best acquire embryos at the following stages: E8.5, 9.0, and 9.5. Embryos were individually flash-frozen and genotyping performed on yolk sac DNA from each sample as described in section 3.4.2. Embryos from the same litter of the same genotype were combined and RNA extracted. A minimum of 4 embryos of each genotype from the same litter was used in the analysis. Refer to section 2.4.8 for procedure of

RNA extraction and making cDNA, with one modification. The tissue for these

RNA extractions was homogenized by use of 0.1 mm diameter glass disruption beads (Scientific Industries) via repeated vortexing of sample and beads in

TRIzol. 500 ng of cDNA was used in all RT-PCR reactions, and were performed twice. See Table 3.1 for primers used.

67 3.4.7 Real-Time PCR

Refer to section 2.4.9 for procedure for real-time PCR. See Table 3.1 for primers used; primer name indicates cDNA being amplified.

Table 3.1 Primer sequences.

Figure 3.1 Retinoic acid synthesis and degradation. Oxidation of retinol into retinaldehyde is the rate limiting step and is reversible and is performed by ADH4. RALDHs convert retinaldehyde into biologically active atRA. Cyp26A1, B1, and C1 degrade retinoic acid into biologically inactive product. Red arrows indicate paths that result in less RA available, while green arrows indicate paths that increase total RA available.

Figure 3.2 Retinoid signaling. A. Retinoic acid receptor (RAR) α, β, and γ and retinoid X receptor (RXR) α, β, and γ are bound to response elements (RARE and RXRE) in the DNA as either heterodimers or homodimers, and result in gene repression. B. Upon binding to RA, RARs and RXRs undergo a conformational change resulting in gene transcription. C. The corepressor TGIF binds to RXRE instead of RXR and gene transcription is repressed. D. Heterodimer is bound in a reverse orientation compared to B and is associated with constitutive gene repression.

Figure 3.3 Retinoid signaling level in Smad2ΔE9,10/+ and wild-type embryos. A. Graphical representation of the TGII allele [112]. B. Wild-type and Smad2ΔE9,10/+ E9.5 littermate embryos displaying darker staining intensity in the Smad2ΔE9,10/+. C. Wild-type and Smad2ΔE9,10/+ E10.5 embryos. Wild-type embryos stain with average intensity, while Smad2ΔE9,10/+ littermate embryos display darker (left), average (middle), and lighter (right) staining intensity. Embryo staining intensity is indicated by the grey letters next to the embryo. L is ligher, A is average, and D is darker. Average intensity is determined uniquely in the individual litter by the staining intersity of the majority of the wild-type embryos, and all embryos within the litter are scored relative to this average. D-E. Charts indicating proportion of embryos from each genotype that visually displayed lighter, average, or darker staining compared to staining intensity of the majority of wild-type embryos within the litter at E9.5 (D) and E10.5 (E).

Figure 3.3

Figure 3.4 Retinoic acid induces HPE in Smad2 embryos. A-B. Charts summarize embryo HPE penetrance in wild-type compared to Smad2ΔE9,10/+ (A) and Smad23loxP/+ compared to Smad23loxP/3loxP (B). C. Representative E9.5 embryos displaying HPE phenotypes.

Figure 3.5 Expression level of RARs and RXRs. Semi-quantitative RT-PCR was performed on embryonic stages 8.5, 9.0, and 9.5. No detectable changes in the RARs or RXRs are observed. Sm2Δ/+ refers to both Smad2ΔE9,10/+ and Smad2ΔC/+ and + is wild-type.

Figure 3.6 Expression level of RA metabolizing genes. Semi-quantitative RT- PCR was performed on embryonic stages 8.5, 9.0, and 9.5. Only changes in ADH4 and Cyp26A1 were detectable. This change is specific to the R-Smad Smad2, as similar changes were not observed with Smad3 on an inbred C57BL/6 strain. Sm2Δ/+ refers to both Smad2ΔE9,10/+ and Smad2ΔC/+, Sm3+/- is Smad3+/-, and + is wild-type.

Figure 3.7 Real-time PCR of ADH4 in Smad2 embryos. Charts summarize ADH4 expression levels comparing wild-type to Smad2ΔE9,10/+ (A) and Smad23loxP/+ to Smad23loxP/3loxP (B) in E8.5 embryos. The genotype with the highest expression level in A and B was considered 100%.

Figure 3.8 ADH4 promoter sequence. Two portions of the ADH4 promoter have tandem repeats of the SBE, in yellow. Red indicates potential RAR and RXR binding sites.

Figure 3.9 Cyp26A1 promoter sequence. Two portions of the Cyp26A1 promoter have tandem repeats of the SBE, in yellow. Red indicates potential RAR and RXR binding sites, and is a DR5 site.

CHAPTER 4

SMAD2 AND TGIF MUTATIONS, IN ADDITION TO STRAIN BACKGROUND,

WORK IN CONCERT TO CAUSE MURINE HOLOPROSENCEPHALY

4.1 Introduction

5’ TG 3’ interacting factor (TGIF) is an atypical homeodomain protein belonging to the TALE (three amino acid loop extension) subfamily of homeodomain proteins [118]. TGIF has been implicated as a transcriptional repressor of both retinoid and transforming growth factor beta (TGF-β) signaling pathways [118, 119]. Mutations in TGIF have also been associated with the human disorder holoprosencephaly [14, 120], a common human developmental defect that affects 1 out of every 250 conceptions, resulting from the failure of the prosencephalon and embryonic eye field to completely separate (reviewed in

[121]).

4.2 Results

4.2.1 Expression Domain of Tgif and Tgif2

In order to further elucidate the role and expression domains of Tgif, expression during early murine embryonic development was analyzed. Previous

80 work to characterize the Tgif expression domains dealt mainly with later

embryonic stages, specifically from embryonic day 12.5 to postnatal, and

Northern blots failed to detect Tgif gene expression any earlier than E9.5 [122].

Recent work on the expression of Tgif also did not analyze stages earlier than

E9.5, and focused mostly with the forebrain at later stages [123]. Humans and

mice both have genes that encode a protein with a high degree of similarity to

Tgif, termed Tgif2 [124]. Tgif2 has been identified as early as E7.5 by reverse

transcription PCR, and in specific tissues in E12.5 and E15.5 mouse embryos

[125]. However, the expression domain of Tgif2 during early stages of

development has not been examined.

Using whole mount in situ procedures with a digoxigenin (DIG)-labeled

antisense probe, it was possible to observe Tgif expression as early as

embryonic day 7.5. Embryos at this stage exhibit expression of Tgif throughout

the embryonic ectoderm, neural ectoderm, and mesoderm, although expression

is largely absent from the extraembryonic membranes (Fig. 4.1A-B). By E8.5-9.0

embryonic Tgif expression is somewhat more restricted, with areas of high

expression localized to the neural folds and gut, while expression appears absent

from the heart and hepatogenic endoderm (Fig. 4.1C-E). A day later in

development, Tgif is highly expressed in the maxillary and mandibular

components of the first branchial arch, the second branchial arch, and the otic

vesicles (Fig. 4.1F). Faint expression is observed in the somites, forelimb buds,

and the gut, while the optic stalks are labeled in the head. Furthermore,

expression is detected in a dorsal anterior domain that may be part of the

81 cephalic mesenchyme. Embryonic day 10.5 embryos continue Tgif expression in

the somites, otic vesicles, and the branchial arches (Fig. 4.1G). In addition, the

tissue of the cephalic mesenchyme appears to also be labeled, expression in the

fore- and hindlimb buds is localized to the apical ectodermal ridge, and

expression in the medial and lateral nasal processes and optic region are

observed. Lastly, at E11.5, Tgif is expressed in the limb buds (Fig. 4.1H-I).

Expression can also be detected in the somites, spinal cord, lateral nasal

process, eyes, and the telencephalic vesicle. Tgif full-length sense probe failed

to hybridize in analyzed embryos (data not shown). Thus Tgif is expressed in the

correct tissues and embryonic stages that are critical for normal craniofacial and

forebrain development.

TGIF2, closely related to TGIF, is also a homeodomain protein that

functions as a transcriptional repressor [125]. Though TGIF2 is 46% similar to

TGIF (data no shown), the mRNA expression domains and temporal expression

differ. Tgif2 expression was not detected in wild-type E9.5 embryos (Fig. 4.2A),

but was detected a day later. Expression at this later stage was localized in the

forebrain within the telencephalic vesicle, the hindbrain, and the heart (Fig.

4.2Cii). Tgif2 is also detected in the branchial arches. There is no Tgif2

expression in the somites, cephalic mesenchyme, or apical ectodermal ridge, all

of which expressed Tgif (Fig. 4.2Ci). Thus, the expression domain of Tgif2 has

overlap and distinct domains to the Tgif expression domains. Mice that completely lack Tgif are viable, healthy, and fertile when maintained on our outbred stock (data not shown). One possible reason for survival may be

82 functional redundancy with TGIF2. To addresses this hypothesis, Tgif2

expression was examined in Tgif -/- embryos (mice were a kind gift from D.

Wotton). What was observed was that Tgif2 expression began at E9.5 in the

Tgif-/- embryos, a day earlier than in the wild-type (Fig. 4.2B). Tgif2 is observed

to be expressed in regions where Tgif would normally be expressed, namely the

branchial arches, the otic vesicles, gut, and optic stalks. Thus, Tgif2 has ectopic

expression in Tgif -/- embryos, so TGIF2 may be able to compensate for the

removal of TGIF.

4.2.2 Retinoid Signaling Affected in Tgif Embryos

Retinoid signaling levels can be observed using an RARE-LacZ reporter

mouse line (TGII) [112]. This line was bred into the Tgif mice and the embryos

were analyzed. Consistent with a role in repressing retinoid signaling, removal of

one or both copies of Tgif resulted in a trend of increased staining, and thus,

increased retinoid signaling. Figure 4.3 A-D show examples of average and

darker staining intensity. No embryo was ever observed staining lighter (Fig.

4.3C). This is different from what was observed in Smad2ΔE9,10/+ embryos, which displayed increases and decreases in retinoid signaling levels (section 3.2.1).

The domain of staining did not change, so retinoid signaling is not occurring in ectopic locations. Recently, it has been observed that embryos harboring one or two copies of Tgif are very sensitive to retinoic acid teratogensis (S. Lasse,

honors thesis and [126]).

83 4.2.3 Frequency and Penetrance of Holoprosencephaly in Smad2/Tgif

Embryos

Since TGIF was associated with human HPE, and was a corepressor of both retinoid signaling and TGF-β signaling (via Smad2), Tgif mutant mice were bred with Smad2 mutant mice to determine if reduction in TGF-β signaling and increases in retinoid signaling have an effect on development. It was observed that when compound heterozygotes were made of Smad2 and Tgif, there was a

40% lethality of the embryos. The major phenotype observed was HPE (Fig 4.4),

and was detected in 30% of the Smad2/Tgif compound heterozygous embryos

(Table 4.1A) (T. Carrel, Ph.D. Dissertation). No HPE was observed in

Smad3/Tgif compound heterozygotes (S. Lasse, honors thesis). However, as

the project continued and bred away from the original founder, an animal on a

mixed C57BL/6 strain background, the penetrance of all phenotypes decreased,

until no phenotypes were ever observed and was considered permanently lost

(data not shown).

Strain background can influence the penetrance and variability of

phenotypes. The agnathia-holoprosencephaly complex phenotype of Otx2

completely depends on strain background and is observed only on C57BL/6

[127]. Valporate-induced teratogenesis is also dependent upon strain

background [128]. Outbred stock lines are bred for maximum heterozygosity, but

are subject to random genetic drift, so the true extent of variation in any particular

individual is not known. In situations where the effect of a drug is being

researched, outbred stocks tend to fail to reproducibly detect the effect (the

84 power of the experiment) because the difference between groups is less than the

differences within the group [129]. Cdo-/- embryos on a 129/Sv background

display mild HPE phenotypes in 50% of the embryos. When bred into the

C57BL/6 background, phenotypic penetrance was above 80%, and the animals displayed more severe HPE and cebocephaly [130]. Finally, the penetrance of the spectrum of brain developmental defects including exencephaly and HPE

that was observed in the heterozygotes and homozygotes of another Tgif mutant

allele, TgifΔexon3, increases at the proportion of C57BL/6 increases [131]. Based

on these reports, and the fact that both TGF-β signaling and retinoid signaling

can depend on strain background, Smad2ΔE9,10/+; Tgif +/- mice were bred near-

congenic into a pure background to attempt to recover the HPE phenotype. In

view of the fact that the C57BL/6 strain appears to be sensitive to anterior

defects like HPE, and that the Tgif founder mouse was on a mixed C57BL/6

background, C57BL/6 was selected for the new background. Briefly, the

breeding scheme is summarized in Figure 4.5, where two brothers congenic on

C57BL/6 acquired from Jax Laboratories were bred to every generation of

Smad2ΔE9,10/+; Tgif +/- females. The percentages indicate the percentage of

C57BL/6 background in each generation and the starting Smad2ΔE9,10/+; Tgif +/- mice were assumed to not retain any C57BL/6 due to the large extent of outbreeding that had occurred. This inbreeding resulted in the final F5 generation

that was, on average, 96.9% C57BL/6.

During the breeding, a slight reduction was observed in the live born

ΔE9,10/+ +/- Smad2 ; Tgif by the F4 generation (data not shown), but breeding

85 continued one more generation. When the F5+1 mice were interbred, there was a

33% partially-penetrant anterior and midline defect phenotypes, the majority of

which was HPE (Fig. 4.6) (Table 4.1B), which is very similar to the original

penetrance level. Thus, it is the combination of reductions in both Smad2 and

TGIF, in addition to an unknown modifier(s) present in the C57BL/6 background

that is not present in the outbred stock of this lab’s mouse colony, which resulted

in a partially-penetrant HPE phenotype.

This unknown modifier(s) does have an effect on Smad2 function.

Previously (section 2.2.2) it was observed that Smad23loxP/ΔE9,10 embryos

displayed phenotypes that could be organized into three main classes [75].

When the analysis was done on the original outbred stock, nearly 30% of the

3loxP/ΔE9,10 embryos could be grouped into Class I. Smad2 F5+3 embryos display

only one phenotype, which is the Class I phenotype (n=13). Previously,

Smad2ΔE9,10/ΔE9,10 embryos were lethal before E8.5 due to gastrulation defects

ΔE9,10/ΔE9,10 (section 2.2.1) [75]. Smad2 F5+3 embryos display two distinct phenotypes (n=10). The early gastrulation defect that results in very small compact embryos was observed in 25% of the null embryos (Fig. 4.7Ai-ii). The remaining 75% of null embryos display a phenotype of a large hollow spherical mass, which can be observed as late as E9.5, which appears to phenocopy

Smad2Robm1/Robm1, Smad2m1Mag/m1Mag, Smad2m1Mag/Robm1, and Smad23loxP/ΔE9,10

Class I embryos [55, 67, 75] (Fig. 4.7Aiii and B). It would appear that the

3loxP/ΔE9,10 Smad2 F5+3 embryos are displaying the more severe Class I defect,

ΔE9,10/ΔE9,10 though Smad2 F5+3 embryos appear to be displaying a milder

86 phenotype compared to what was originally observed. Perhaps, when

considering Smad2 removal or reduction below a threshold, the empty yolk sac

phenotype is the most severe that can be displayed on the C57BL/6 background.

As such, Smad2ΔE9,10/ΔE9,10 and Smad23loxP/ΔE9,10 phenotypes shifted to be the

most severe phenotypes. The recovery of some gastrulation defects in

ΔE9,10/ΔE9,10 Smad2 F5+3 embryos may be due to less C57BL/6 being present in

those particular embryos compared to others (since the background is not fully

3loxP/ΔE9,10 congenic), and if more Smad2 F5+3 embryos are collected perhaps a

few will be of either Class II or Class III. That such embryos were not observed

could be by random chance.

Finally, when semi-quantitative RT-PCR for ADH4 and Cyp26A1 was

performed on F5+3 E8.5 embryos, a difference was observed compared to what

was originally detected. Whereas before both ADH4 and Cyp26A1 were reduced

at this early stage in the Smad2ΔE9,10/+ outbred embryos, now only ADH4

reduction can be detected (Fig. 4.7C).

4.2.4 Molecular Analysis of Smad2/Tgif Embryos

To investigate the possible molecular mechanisms underlying the

sensitivity of Smad2/Tgif F5+3 compound heterozygous embryos to HPE, genes

involved in retinoid signaling were analyzed via semi-quantitative RT-PCR at

E8.5 (Fig. 4.8). Emphasis was placed on genes whose transcription level was affected in Smad2ΔE9,10/+ embryos to determine if there were any differences.

ΔE9,10/+ ADH4 transcription level was still reduced in Smad2 F5+3 embryos 87 +/- ΔE9,10/+ compared to wild-type or Tgif F5+3 littermates. Surprisingly, in Smad2 ;

+/- Tgif F5+3 embryos, ADH4 expression level is comparable to wild-type levels.

Thus, it would appear that removal of an allele of Tgif is associated with ADH4

expression, reversing the effect of removing one allele of Smad2 through an

ΔE9,10/+ unknown mechanism. Cyp26A1 expression is detectable in Smad2 F5+3

embryos, which was not what was observed previously (Fig. 3.6). Embryos that have one allele of Tgif removed display a reduction in Cyp26A1 expression level;

+/- ΔE9,10/+ +/- this can be observed in both Tgif F5+3 and Smad2 ; Tgif F5+3 embryos.

This may be one contributing factor as to why retinoid signaling is increased in

Tgif mutant embryos; reduction in Cyp26A1 should result in elevated levels of

atRA because it can no longer be quickly degraded [101], and thus, results in increased retinoid signaling. Additionally Shh, a target of RA involved in ventral

neural tube development and linked to HPE, was observed to be reduced in all

transgenic embryos at E8.5 (Fig. 4.8). Shh repression detected in the Tgif

mutant embryos has also been observed previously and believed to be due to

increased retinoid signaling in these mutant embryos [131]. This is consistent

with increased retinoid signaling, as RA results in repression of Shh [132].

Reduction of Shh detected in the Smad2ΔE9,10/+; Tgif+/- embryos is further

confirmation that retinoid signaling is elevated.

ΔE9,10/+ +/- One gene of interest that was reduced only in Smad2 ; Tgif F5+3

embryos was Cyp26C1. Like Cyp26A1, Cyp26C1 is an enzyme that can

degrade retinoic acid [133]. The reduction of Cyp26A1 and Cyp26C1 in

Smad2ΔE9,10/+; Tgif +/- embryos may be a large contributor to the HPE phenotype

88 observed in some of these embryos. Unlike Cyp26A1-/- embryos, Cyp26C1-/- mice have no phenotype [134], possibly due to redundancy with other Cyp26 family members. However, when Cyp26A1-/-; Cyp26C1-/- embryos were

observed, there were completely penetrant CNS abnormalities that affected

anterior development [134]. Thus, reduction in both these degrading enzymes in

ΔE9,10/+ +/- Smad2 ; Tgif F5+3 embryos may result in a situation where an inability to

degrade and remove the atRA occurs. This could result in a local increase in

atRA to teratogenic levels that cannot be overcome in a proportion of these

embryos, and anterior defects including HPE occur. Alternatively, the embryo

samples that were used for semi-quantitaitve RT-PCR were combined, and thus,

are averages of affected gene transcription. Further analysis will need to be

done to determine if in every Smad2/Tgif F5+3 mutant embryo Cyp26A1 and

Cyp26C1 expression is reduced. Unlike in Cyp26A1 gene expression, Smad2

and TGIF are synergistically having an effect upon Cyp26C1 expression.

4.2.5 Sensitivity of Smad2/Tgif Embryos to Retinoic Acid Tertaogensis

Both Tgif +/- and Smad2ΔE9,10/+ embryos are sensitive to teratogenic in utero exposure to atRA [126] and Figure 3.4. Preliminary data seem to indicate

ΔE9,10/+ +/- that Smad2 ; Tgif F5+3 embryos were more sensitive to teratogenic levels of atRA compared to the wild-type and heterozygous littermates. However, not

ΔE9,10/+ +/- enough data exists to determine if the sensitivity of Smad2 ; Tgif F5+3

embryos to atRA is additive or synergistic. What is observed is that Smad2/Tgif

F5+3 embryos display more severe phenotypes when compared to their

89 littermates (Fig. 4.9). The wild-type embryo lacks only the right eye, while the

ΔE9,10/+ Smad2 F5+3 embryo displays HPE, cebocephaly, and lacks a clearly-

+/- defined eye structure. The Tgif F5+3 embryo displays smaller than average eye

ΔE9,10/+ +/- size and a mild cleft palate, as compared to the Smad2 ; Tgif F5+3

embryo, which displays anterior truncation, the most severe phenotype of all the littermates. Thus, reduction in Tgif or Smad2 sensitizes the embryo to

teratogenesis via atRA, while reductions in both alleles could result in greater

sensitivity to increasingly severe phenotypes to the same dose of atRA.

4.3 Conclusions

Mutations in TGIF, an atypical homeodomain protein, have been

associated with the human disorder holoprosencephaly [14, 118, 120]. However,

Tgif null mice fail to display HPE (data not shown). The mRNA expression domain of Tgif in early embryonic development had been determined, and is present in structures important for correct craniofacial and forebrain development, like the branchial arches and the neural folds. Tgif2 shares similarity to Tgif structurally, but does not display identical expression domains.

Tgif2 is absent in the somites, cephalic mesenchyme, and apical ectodermal ridge, all of which express Tgif. Additionally, Tgif is expressed at E9.5, when

Tgif2 expression is absent. TGIF2 may functionally compensate for TGIF in

Tgif -/- embryos, as it was observed that Tgif2 expression was present in E9.5

Tgif -/- embryos and could be a contributing factor as to why Tgif null embryos are

able to develop normally. TGIF is a transcriptional repressor of both retinoid and

90 transforming growth factor beta (TGF-β) signaling pathways [118, 119].

Consistent with a role in repressing retinoid signaling, removal of one or both

copies of Tgif resulted in increased retinoid signaling (S. Lasse, honors thesis)

and increased sensitivity to atRA teratogenesis (S. Lasse, honors thesis) [126].

Previous work in the lab created compound heterozygotes of Smad2 and Tgif,

resulting in a 40% lethality of the embryos and the major phenotype observed

was HPE (T. Carrel, Ph.D. Dissertation). However, as the project continued and

bred away from the original founder, the phenotypes were considered lost.

Strain background can play a major role in the penetrance of phenotypes,

and can modify and influence signaling pathways [127, 128, 129, 130, 131]. Our

data indicate that strain background is fundamentally important to the penetrance

of the HPE phenotype in Smad2ΔE9,10/+; Tgif +/- embryos. Smad2/Tgif compound

heterozygous mutant mice were bred 5 generations to pure C57BL/6 mice, then

ΔE9,10/+ +/- interbred. The Smad2 ; Tgif F5+3 embryos display the HPE phenotype.

One of the goals of mouse research is to help understand human development, diseases, and disorders. As such, the closer the mouse can model the human, possibly the more relevant the results will be. HPE is a complex disorder, and though mutations in a number of genes have been associated with patients, this explains less than 30% of the cases. There is, therefore, a multiple- hit theory for human HPE, in which unknown modifiers, other unknown genes, and environmental effects may play a role in determining the etiology of the disorder [131]. ADH4 has been associated with mouse embryo sensitivity to fluctuations in RA levels [102], and expression levels were found to be affected in

91 Smad2ΔE9,10/+ embryos. ADH4 has two allozymes, ADH4A and ADH4B, the

difference being due to position 120 in the outer loop of the substrate binding

pocket; ADH4a encodes for a cycteine at that position, while ADH4b encodes an

arginine [135]. ADH4A is the only allozyme present in most laboratory strains of mice, while only ADH4B is present in the C57BL/6 strain [136] and in humans

[137]. The kinetic properties for these two allozymes differ as ADH4B has a lower Km for ethanol than ADH4A does, resulting in less ethanol substrate

required before all ADH4B is saturated in enzyme-substrate complexes [135].

Thus, by breeding into the C57BL/6 mouse strain, the ADH4 allozyme present in

humans will be the same, resulting in our mouse model being more similar to

humans in this regard.

Strain background had a large role in the underlying etiology of the HPE phenotype observed in the Smad2/Tgif compound heterozygous C57BL/6 mutant

mice. Without the unknown modifier(s) present in this strain of mouse compared

to the random mixed outbred stock, no phenotype would have been observed.

Was the C57BL/6 strain completely responsible for the observed phenotype

since its removal, then subsequent re-introduction, coincided with the penetrance

of HPE? Originally, the Smad2/Tgif mice were, at the most, 50% C57BL/6 since

interbreeding into this lab’s outbred stock began immediately. To re-acquire the

HPE phenotype displayed by Smad2/Tgif embryos, the compound heterozygous

mice were bred to C57BL/6. Smad2/Tgif compound heterozygous mice was

observed once the proportion of C57BL/6 was more thsn 93% (data not shown).

There is a possiblity that there are additional factors other than the proportion of

92 C57BL/6 that are involved in the penetrance of HPE. Alternatively, breeding

these mice congenic into C57BL/6 would randomly removed modifier gene(s)

accidentally introduced into the Smad2/Tgif mice from another strain background.

As a heterozygous outbred stock, interbreeding with all mice in the colony did

occur, and as new transgenic mice on different backgrounds were brought into the colony, they bred and mixed into the population. Transgenic mice, for example, that had been isolated and only produced offspring of the agouti coat

color could, a few generations after interbreeding with the general population, produce offspring that were agouti, white, black, brown, and tan colored. Thus it is possible that a transgenic mouse was acquired and bred into the Smad2/Tgif colony, introducing an unknown background strain potentially containing unique modifier(s) that resulted in the loss of the phenotypes. As such, breeding to almost any pure mouse line would have had the same removing effect, and the penetrance of HPE would have eventually increased. These unknown modifier(s) in the background may be acting on TGF-β signaling through Smad2 or TGIF, retinoid signaling via Smad2 or TGIF, on each of these signaling pathways at once, or on another pathway involved in forebrain development.

Previous work has suggested that strain background can affect phenotype penetrance in TgifΔexon3 mice, presumably though the retinoid pathway [132].

Smad23loxP/ΔE9,10 near-congenic C57BL/6 embryos have less variation in

phenotypes, and display the Class I phenotype of an empty yolk sac, while

Smad2 null embryos have been observed to display two phenotypes, which

includes an empty yolk sac phenotype not previously observed with this allele.

93 Cyp26A1, found to be reduced in E8.5 Smad2ΔE9,10/+ embryos (Fig. 3.6), is not

ΔE9,10/+ affected in Smad2 F5+3 embryos (Fig. 4.8). Thus, strain background can

affect many pathways, and can be affecting both TGF-β and retinoid signaling.

Reduction in the gene expression levels of both Cyp26A1 and Cyp26C1 in

Smad2ΔE9,10/+; Tgif +/- embryos may be contributing to the observed HPE and

anterior defects. CNS defects were discerned in Cyp26A1/Cyp26C1 null

embryos [134], and a reduction in both these degrading enzymes in

Smad2ΔE9,10/+; Tgif +/- embryos was observed. Not all Smad2/Tgif embryos

develop abnormally; similarly not all wild-type embryos exposed to teratogenic

substances develop abnormally on pure strain backgrounds [113, 128]. Similar

to human HPE cases, there is no absolute correlation between an affected gene

and the severity of phenotype, if one is even displayed, only an association

(reviewed in [12]). To display the HPE phenotype, other gene product(s) or

environmental factor(s) can be required to act in conjunction with the inherent

sensitivity unique to the individual embryo.

4.4 Materials

4.4.1 Mice and Matings

Wild-type, Tgif+/-, and Tgif-/- mice used for acquiring embryos for whole

mount in situ were maintained on a background which included a random mixture

of 129SvEv, NIH Black Swiss, and C57BL/6. Smad2ΔC/+, Smad2ΔE9,10/+,

Smad23loxP/+, wild-type, Tgif +/-, Smad2ΔC/+; Tgif +/-, and Smad2ΔE9,10/+; Tgif +/- were all bred near-congenic into C57BL/6 to generation F5+3 where stated. F5+3 were

94 generated by mating together mice at generation F5+2, who in turn were derived

from interbreeding mice of the F5+1 generation. Mouse lines not mentioned in this

section but that simultaneously underwent the same procedure for breeding

near-congenic into C57BL/6 were the following: TGII, Smad3+/-, and Smad4Δ/+.

Near-congenic C57BL/6 mice were maintained on a high fat breeder diet. See

section 2.4.3 for specific procedure for matings and acquiring embryos for

analysis. Embryos were stored in 100% MeOH at -20oC until use for whole

mount in situ analysis.

4.4.2 Genotyping

Genotyping of mice was performed by PCR on either tail or toe biopsy,

while genotyping of embryos was on extraembryonic membranes, or embryonic

tissue. For genotyping primers used, see Table 2.2. The following primers sets were used: WT: SM2-16 and R2; Smad2ΔE9,10: SM2-16 and WTR; Smad2ΔC:

Fu-1 and RINA. For alleles specific to this chapter, see Table 4.2. The following primer sets were used: WT: Int-1 and Exo2-5; Tgif-: Int-1 and GFPlow2 and neo:

CTNeo1 and CTNeo2 (Table 2.2). For PCR procedure, see section 2.4.4.

4.4.3 Histology

After whole mount in situ, embryos were perfused in paraffin and sectioned by

standard procedures as described in section 2.4.5 to a thickness of 12 µm with

no counterstaining.

95 4.4.4 Whole Mount In Situ Hybridization

Whole mount in situ hybridization was performed as described [138].

4.4.4.1 Preparation of embryos

Embryos were obtained and fixed as described in section 2.4.3. The

embryos were then washed twice in 1x PBS with 0.1% Tween-20 for 10 minutes

at room temperature. Embryos were dehydration by passing them through a

methanol series (25%, 50%, 75%, 100%), washing in each concentration of alcohol for 1 hr at room temperature. Embryos were then transferred to a 2-ml screw cap tube.

4.4.4.2 Synthesis of the digoxigenin (DIG)-UTP Labeled RNA Probes

In an RNase-free microcentrifuge tube, the following was added in this

order: 1µg of purified linear DNA template, 2 µl rNTP labeling mix containing

10mM rATP, rGTP, rCTP, 3.5 mM rUTP, and 6.5mM DIG-rUTP, 2 µl 10x

transcription buffer, 1 µl RNase inhibitor and 2 µl of the appropriate RNA

polymerase (T3, T7, or Sp6). DepC-treated water (made by standard

procedures) was used to bring the final volume to 20 µl. After mixing well, the

reaction was incubated for two hours at 37oC, then 2 µl of DNase I was added

and the reaction was incubated for 15 minutes longer. To precipitate the probe,

2 µl 0.5 M EDTA was added, and the volume of the reaction was brought to 50 µl

with DepC water, followed by the addition of 5 µl of 4 M LiCl, and 130 µl of 100%

96 ethanol. Following incubation at -80oC for 30 minutes, the reaction was spun in

the microcentrifuge. The pellet was then washed twice with 70% ethanol in

DepC water and dissolved in 20 µl of DepC water.

4.4.4.3 Hybridization of DIG-Labeled Probes to Embryos

To remove any endogenous RNase activity, the embryos were washed for

two hours in 5% hydrogen peroxide in methanol, then rehydrated by passing

them through a methanol series (100%, 75%, 50%, 25%) for 1 hour each. Next,

the embryos were rinsed twice, for 10 minutes each, in 1x PBTX (PBS, 0.1%

Tween-20). The embryos were then incubated in 2 ml of 10 µg/ml proteinase K

in PBTX, approximately 7 minutes for E7.5 up to 15 minutes for E12.5 embryos.

Following this incubation, the embryos were washed twice in PBTX for 10

minutes each. To ensure complete fixation and to hold the embryo together, the

embryos were fixed again (1x PBST, 0.2% glutaraldehyde, 4% PFA) at room temperature for 20 minutes. The fixative solution was washed off by PBTX for 10 minutes, two times. Hybridization solution was then added (50% formamide, 5x

SSC, 2% Blocking Powder, 0.1% Tween-20, 0.5% CHAPS, 100 µg/ml of yeast tRNA, 5mM EDTA and 50 µg/ml heparin). The tubes were incubated for 2 hours at 65oC. After this, fresh 2 ml of pre-warmed hybridization buffer containing the

labeled riboprobe was added to the tube. The embryos were then incubated

overnight at 65oC. The embryos were washed at 65oC for 5 minutes in 100%

Solution I (50% formamide, 5x SSC, 0.1% Tween-20, and 0.5% CHAPS),

followed by 75% Solution I / 25% 2x SSC, 50% Solution I / 50% 2x SSC, and

97 25% Solution I / 75% 2x SSC. The embryos were then washed for 30 minutes

twice in 2x SSC, 0.1% CHAPS, then 0.2x SSC, 0.1% CHAPS, at 65oC.

Afterward, the embryos were washed twice for 10 minutes each in TBTX (50 mM

Tris-HCl pH 7.5, 150mM NaCl, and 0.1% Tween-20) at room temperature.

4.4.4.4 Anti-DIG antibody (AP-conjugated) Binding

The embryos were pre-blocked for non-specific binding in 2% BSA in

TBTX for 3 hours at room temperature. Meanwhile, the pre-absorbed anti-DIG antibody was prepared by mixing 3 mg of embryo powder (ground dehydrated embryos at the same stages of the embryos going through the whole mount procedure) and 0.5ml 1x TBTX. This was incubated at 70oC for 30 minutes, vortexed, then allowed to cool on ice. 1 µl of anti-DIG-AP FAb (Boehringer) was added, along with 5 µl of goat serum, and antibody was allowed to preabsorb for

1 hour with gentle rocking at 4oC. Afterward, the mixture was spun in a

microcentrifuge for 10 minutes at 4oC. Finally, the supernatant was diluted into

2ml of 10% goat serum, 2% BSA. After the embryos have been pre-blocked, the blocking solution was removed, and the pre-absorbed antibody solution was added. The anti-DIG antibody was incubated with the embryos at 4oC, overnight.

The embryos were then washed 5 times, in TBTX containing 0.1% BSA for 1

hour each at room temperature. A final overnight wash was done at 4oC.

98 4.4.4.5 Immunohistochemistry

The embryos were washed twice in TBTX containing 2mM levamisole for

15 minutes at room temperature, followed by NTMT (100 mM NaCl, 100mM Tris-

HCl pH 9.5, 50mM MgCl2 and 0.1% Tween-20) containing 2 mM levamisole,

three times for 10 minutes each at room temperature. After the washes, 2 ml of

AP substrate containing 6.75 µl NBT (Nitro-Blue Tetrazolium Chloride) and 5.25

µl BCIP (5-Bromo-4-Chloro-3'-Indolyphosphate p-Toluidine Salt) (Boehringer) in

NTMT with 2mM levamisole was added to each tube containing the embryos.

The tubes were immediately wrapped in aluminum foil to create a dark environment, and rocked until the color reaction was complete at room temperature or 4oC. The color reaction was stopped by washing in NTMT,

followed by washes with PBTX. A final fixation in 0.2% glutaraldehyde, 4% PFA

preserved the embryos for photographing. Embryos were either dehydrated in a

methanol series for sectioning, or stored at -20oC in 100% MEOH.

4.4.5 Teratogenic Application of Retinoic Acid

Timed mating was set up as described in section 2.4.3. To acquire

ΔE9,10/+ +/- ΔE9,10/+ +/- Smad2 ; Tgif embryos, Smad2 ; Tgif F5+3 males were paired with

wild-type F5+3 females, all of similar age and weight. Retinoic acid was administered as described in section 3.4.3, with the exception that the atRA dose

was 3-4 mg per kg of maternal weight.

99 4.4.6 RNA Extraction and Analysis

Timed mating were set up as described in section 2.4.3. To acquire

ΔE9,10/+ +/- embryos, Smad2 ; Tgif F5+3 males were paired with wild-type F5+3 females. Dissections were performed in the mornings to best acquire E8.5 embryos. Refer to section 3.4.6 for RNA extraction and cDNA procedure. See

Table 2.2 for primers used in the following sets: HPRT: HPRT L and HPRT R.

See table 3.1 for primer sequences for RA metabolizing genes. Sequences specific to this chapter can be found in Table 4.2. The following primer sets were used: Cyp26C1: Cyp26C1 F and Cyp26C1 R and Shh: Shh F and Shh R.

100

Table 4.1 Intercrosses between Smad2 and Tgif result in partially-penetrant HPE. Genotypic analysis of offspring resulting from crosses between mice harboring Smad2 and Tgif mutant alleles and the percentage of embryos displaying HPE. A. HPE is displayed in 30% of the Smad2/Tgif heterozygous embryos at E10.5 on a mixed strain background (T. Carrel, Ph.D. Dissertation). Parental mice were of mixed genotypes of Smad2 and/or Tgif, and this table is a summary of all these matings. B. HPE is displayed in 24% of Smad2/Tgif heterozygous embryos at E9.5-11.5 on a near-congenic C57BL/6 strain background.

101

Table 4.2 Primer sequences.

102

Figure 4.1 Expression of Tgif in early-stage mouse embryos. Tgif expression during A-B embryonic day 7.5, C-E day 8.5-9.0, F E9.5, G E10.5, and H-I E11.5. A, C,and E. Whole mount in situ embryos labeled with a DIG-labeled antisense probe. B and D. Sections of whole mount in situ embryos with no counterstain. Tgif is expressed in the stages and tissues involved in correct craniofacial and neural patterning. Abbreviations: aer-apical ectodermal ridge, b-second branchial arch, cm-cephalic mesenchyme, e-endoderm (A), e-eye (H), f-forelimb, g-gut, h-heart, m-maxillary and mandibular components of the first branchial arch (F, G), m-mesoderm (B), nc-neural crest, ne-neural endoderm, nf-neural folds, np-nasal process, os-optic stalk, o-otic vesicle, s-somites, sc-spinal cord, t- telencephalon.

103

Figure 4.1

104

Figure 4.2 Expression of Tgif2 during mouse embryogenesis. Tgif2 expression during A-B E9.5 and Cii E10. A. Tgif2 is not expressed in wild-type E9.5 embryos, but is expressed in Tgif -/- embryos (B). C. Tgif2 is expressed in wild- type E10.5 embryos (ii) in unique domains compared to Tgif expression (i). Abbreviations: aer-apical ectodermal ridge, cm-cephalic mesenchyme, fb- forebrain, h-heart, m-maxillary and mandibular components of the first branchial arch, s-somites.

105

Figure 4.3 Retinoid signaling level in Tgif mutant embryos. A. Wild-type, B and C. Tgif +/-, and D. Tgif -/- E9.5-10.0 embryos displaying average and darker staining intensity. E. Chart indicating proportion of embryos from each genotype that visually displayed lighter, average, or darker staining compared to staining intensity of the majority of wild-type embryo littermates. Embryo staining intensity is indicated by the grey letters next to the embryo. L is ligher, A is average, and D is darker.

106

Figure 4.4 Holoprosencephaly phenotype. HPE is observed in a portion of Smad2ΔC/+; Tgif +/- E10.5 embryos on an outbred background. Arrows point to each hemisphere of the forebrain.

107

Figure 4.5 Breeding scheme for Smad2/Tgif mutant mice. Smad2/Tgif mutant females on the outbred background (generation F0) were bred to two pure C57BL/6 sibling males. All Smad2/Tgif mutant female descendants would breed back to the original pure C57BL/6 mice. Percentages indicate average percentage of C57BL/6 in the mouse. Once F5 generation was obtained, mice were interbred.

108

Figure 4.6 Holoprosencephaly phenotype. HPE is observed in a portion of Smad2ΔC/+; Tgif +/- embryos on a near-congenic C57BL/6 background. A. E10.5 embryo displaying HPE. B. E11.5 embryo displays a mild HPE, cebocephaly, and a single eye located on the left side of the head. Frontal (i) and side (ii) views of the same embryo.

109

Figure 4.7 Affects of Smad2 on a near-congenic C57BL/6 background. A. Smad2Δ/Δ embryos at E8.5 display gastrulation defects (i and ii) as previously described [56, 75], and a large empty yolk sac structure that can be observed as late as E9.5 (Aiii and B). C. Semi-quantitative RT-PCR of E8.5 embryos reveal only ADH4 gene expression in reduced in Smad2 heterozygotes. Smad2Δ/+ refers to both Smad2ΔE9,10 and Smad2ΔC alleles, + is wild-type.

110

Figure 4.8 Expression level of RA metabolizing genes. Semi-quantitative RT- PCR was performed on E8.5 F5+3 C57BL/6 embryos. Reduction in Cyp26A1 was observed in embryos with one mutant Tgif allele, regardless of Smad2 mutation. Reduction in Cyp26C1 was only detected in Smad2ΔE9,10/+; Tgif +/- embryos, indicating synergistic regulation. Changes in ADH4 were only observed in Smad2ΔE9,10/+ embryos, which was reversed in Smad2ΔE9,10/+; Tgif +/- embryos. The RA target Shh is also affected in all embryos but wild-type (+).

111

Figure 4.9 Severity of phenotype observed in Smad2/Tgif embryos exposed to atRA. Embryos were exposed to atRA and analyzed at E16.5. All embryos display phenotypes (left), but the Smad2ΔE9,10/+; Tgif +/- embryo is the most severe with anterior truncations. Fetal brain of the corresponding embryo is on the right.

112

CHAPTER 5

SMAD2 AND CYP26A1 MUTATIONS WORK IN CONCERT TO CAUSE

MURINE HOLOPROSENCEPHALY

5.1 Introduction

Embryos can regulate the level of retinoic acid to which cells and tissues are exposed by controlling where RA is synthesized and removing RA to sub- threshold levels where it is not required. This control results in confining RA to the tissue where it is needed. One family of enzymes involved in catabolism of many substrates are the cytochrome P450 enzymes, and atRA is specifically catabolized by cytochrome P450RAI-1 (Cyp26A1) [139], cytochrome P450RAI-2

(Cyp26B1) [140], and Cyp26C1 [141], all encoded by separate genes. Cyp26A1 is first expressed in development around E8.5 in the tail bud neural plate, the hindgut endoderm, and mesoderm [142, 143]. Regulation of Cyp26A1 occurs at the level of transcription involving a retinoid auto-regulation feedback loop;

113 consequently, upon exposure to vitamin A or retinoic acid, transcription increases

[144]. When RA levels fall below a threshold, Cyp26A1 transcription drops to

basal levels.

Complete removal of Cyp26A1 in the mouse results in phenotypes similar

to maternal oral exposure of teratogenic levels of retinoic acid at E8.5-12.5 [145,

146]. Such phenotypes include exencephaly, spina bifida, posterior body

truncations and hindlimb fusion (sirenomelia, or the mermaid syndrome in

humans) [101]. The underlying etiology of these posterior phenotypes in

Cyp26A1-/- embryos is believed to be due to premature RA diffusion from the

trunk of the embryo into the tail bud, which can no longer quickly degrade and clear the RA [101].

However, even though the posterior portion of the embryo is affected, the

anterior portion remained, for the vast majority of the embryos, normal. Cyp26A1

is normally expressed in tissue that should give rise to the forebrain and midbrain

[82, 142]; this pattern disappears before somitogenesis and Cyp26A1 is then

expressed in rhombomere (r) 2 in the hindbrain [101]. In Cyp26A1-/- embryos, the overall organization of the brain is not exceedingly abnormal. The rhombomeres are spacially organized normaly, though r4 is slightly expanded in size and parts of r2 and r3 may be undergoing a partial transformation into an r4- like rhombomere [101]. However, the authors proposed that complete removal of

Cyp26A1, though this should increase the amount of endogenous RA, resulted in

114 high levels of RA only in a very restricted portion of the hindbrain. Therefore, teratogenic levels of RA were not present in throughout the CNS, which resulted in grossly normal development.

There is another Cyp26 expressed in the early brain region - Cyp26C1

[134]. Like Cyp26A1, Cyp26C1 can catabolize atRA [133]. Additionally, unlike

Cyp26A1-/- embryos, Cyp26C1-/- mice do not exhibit phenotypic abnormalities

[134], perhaps due to redundancy with other Cyp26 family members that can also catabolize RA. However, when Cyp26A1-/-; Cyp26C1-/- embryos were analyzed, researchers identified completely penetrant anterior phenotypes that

resulted in lethality by E11.0 [134]. These anterior phenotypes were reminiscentH of exposure to teratogenic levels of RA, Thus, removal of both Cyp26 family members resulted in increased retinoid signaling early in development [134], leading to anterior phenotypes not observed in the individual knockout embryos.

5.2 Results

5.2.1 Frequency and Penetrance of Holoprosencephaly in Smad2/Cyp26A1

Embryos

Embryos reduced in Smad2 are sensitive to HPE upon exposure of teratogenic levels of atRA (section 3.2.2), and Smad2ΔE9,10/+ embryos displayed misregulation of retinoid signaling levels (section 3.2.1), thus indicating retinoid signaling is being misregulated. Cyp26A1 transcription levels are also reduced in

Smad2ΔE9,10/+ E8.5 embryos on an outbred stock compared to wild-type littermates (section 3.2.3). In order to genetically recreate elevated levels of

115 endogenous RA, and since Cyp26A1 already appeared to be affected in our mice

Cyp26A1 targeted deletion mice were acquired [101], and bred to the Smad2

heterozygous mice. The Cyp26A1 mice were on a C57BL/6 background, so

were matched to Smad2 heterozygous mice also on a C57BL/6 background.

After the breeding was completed, it was determined that on the C57BL/6

background, Cyp26A1 expression level is not affected in the Smad2ΔE9,10/+ embryos (section 4.2.3). This may explain why Cyp26A1+/-; Smad2ΔE9,10/+

embryos analyzed at E11.5-16.5 revealed no observable phenotype (Table

5.1A), and these mice were born healthy and fertile. A hypothesis was that the

removal of one Cyp26A1 gene combined with the reduction of Cyp26A1 mRNA

observed in Smad2 heterozgotes would result in embryos severely lacking, or

completely devoid, of Cyp26A1. Complete removal of Cyp26A1 was lethal [101],

so we expected Cyp26A1+/-; Smad2ΔE9,10/+ embryos to display a lethal

phenotype. However, on the C57BL/6 background, Cyp26A1 expression level is not affected in Smad2 heterozygotes, so Cyp26A1 expression level would not be reduced in Cyp26A1+/-; Smad2ΔE9,10/+ embryos compared to Cyp26A1+/-embryos,

which are not lethal. Cyp26A1-/-; Smad2ΔE9,10/+ embryos, however, were not

recovered at Mendelian ratios, as 1 embryo was observed when 4 were

expected. Analyzing earlier stages, Cyp26A1-/-; Smad2ΔE9,10/+ embryos could be

identified and displayed anterior defects, 42% displaying HPE (Table 5.1B and

Fig. 5.1). This anterior defect is not observed in the Cyp26A1-/- embryos.

116 5.2.2 Cyp26A1 and the Smad2 Hypomorphic Allele

Though removal of one copy of Cyp26A1 is not associated with lethality, the embryos could still have a slight inability to quickly degrade RA. Thus, such embryos may have slight sensitivity to other fluctuations in RA and retinoid signaling. Smad2ΔE9,10/+ embryos display an inability to appropriately self-

regulate the level of retinoid signaling that is occurring. Nevertheless, this misregulation combined with a reduction in Cyp26A1 is not lethal to the embryos.

With the use of the Smad2 hypomorphic allele (section 2.2.2), it is possible to reduce wild-type Smad2 to levels below what is found in Smad2 null heterozygotes. However, when this breeding was conducted, Smad23loxP/+;

Cyp26A1+/- and Smad23loxP/3loxP; Cyp26A1+/- mice were viable (Table 5.2A),

healthy, and fertile. Smad2 levels could not be reduced more, as the

Smad23loxP/ΔE9,10 genotype by itself is early lethal. Thus, it would appear that

complete removal of Cyp26A1 is required for the HPE phenotype observed in

Cyp26A1-/-; Smad2ΔE9,10/+ embryos, and cannot be substituted with reducing

Smad2 levels in Cyp26A1 heterozygous embryos.

5.2.3 Smad2, Cyp26A1, and Tgif

To result in embryonic lethality, Cyp26A1 must be reduced by more than half [101]. It is interesting that Cyp26A1 expression levels were observed to be

reduced in Tgif +/- E8.5 embryos, and that this reduction appeared to be

enhanced in Smad2ΔE9,10/+; Tgif +/- embryos. (Fig. 4.8). Additionally, Cyp26C1

expression was reduced only in Smad2ΔE9,10/+; Tgif +/- embryos. Considering the

117 effect Tgif has upon Cyp26A1 expression, the reduction of Cyp26C1 expression

level observed in Smad2/Tgif compound heterozygotes, and removal of Cyp26A1

and Cyp26C1 results in anterior phenotypes [134], Cyp26A1/Smad2 mice were

bred to Tgif F5+3 mutant mice to determine if these embryos displayed a

phenotype. The analysis from 7 litters could indicate that there is a partially-

penetrant phenotype only in the Smad2ΔE9,10/+; Cyp26A1+/-; Tgif +/- mice (Table

5.2B, only relevant genotypes shown), as 60% of the mice are not observed at

P21. Due to the partially-penetrant lethality of Smad2ΔE9,10/+; Tgif +/-, this genotype by itself would account for a portion of the compound triple heterozygotes resulting in lethality. Five mice were expected, two were observed at P21, and (an average) 1.5 mice would result in lethality due to the

Smad2ΔE9,10/+; Tgif +/- genotype (which is 33% of the total triple heterozygotes).

The remaining triple compound heterozygote mice (1.5 mice, or 33% of the total)

were unaccounted for. Possibly these sensitized embryos cannot tolerate the

combinations of removal of one Cyp26A1 allele, reduction in Cyp26A1 and

Cyp26C1 transcription that results from Smad2ΔE9,10/+; Tgif +/-, in addition to an increase and misregulation in retinoid signaling resulting from reduction in TGIF and Smad2. More in depth analysis will need to be done to discover if removing

a single copy of Cyp26A1 has an effect, and if the phenotype that the compound

triple heterozygotes display is that of posterior defects seen in Cyp26A1-/-

embryos, the anterior defects seen in Cyp26A1-/-; Cyp26C1-/- embryos, or both.

118 5.3 Conclusions

Removal of Cyp26A1 in murine embryos results in posterior phenotypes

reminiscent of teratogenic exposure to RA in later stages of embryonic

development. Removal of Cyp26C1 in addition to Cyp26A1 results in an earlier

anterior phenotype reminiscent of teratogenic exposure of RA in early stages of

development. Similarly, anterior defects like HPE can be observed in Cyp26A1-/- embryos when Smad2 is reduced by removing one copy of the gene. Since

Smad2 could affect the regulation of RA, and results in misregulation of retinoid signaling, then combined with the complete removal of Cyp26A1, this situation results in an environment predicted to be teratogenic for the developing forebrain. Complete removal of Cyp26A1 is required for this, as the reduction of

Smad2, as would be found in the Smad23loxP/3loxP; Cyp26A1+/-, was not enough to

result in lethality.

Considering the phenotype observed in Smad2ΔE9,10/+; Cyp26A1-/-

embryos was anterior defects, and removal of Cyp26A1 and Cyp26C1 also result

in anterior defects [134], it would be interesting to determine if Cyp26C1

expression is affected in Cyp26A1-/-; Smad2ΔE9,10/+ embryos. Such a reduction

could contribute to the anterior defects observed in the Smad2ΔE9,10/+; Cyp26A1-/-

embryos.

Similar to the analysis with Smad2/Tgif mutant embryos, this analysis

utilizing a Cyp26A1 null allele in combination with a Smad2 null allele provides

119 support to the theory that the effects of reduction in retinoid signaling and TGF-β

signaling can synergistically combine to result in a specific defect –

holoprosencephaly.

5.4 Materials

5.4.1 Mice and Matings

All mice were maintained on a near-congenic C57BL/6 background, and fed a high fat breeder diet. See section 2.4.3 for specific procedure for matings and acquiring embryos for analysis. Briefly, a Smad2ΔE9,10/+; Cyp26A1+/- mouse would be paired with a Cyp26A1+/- mouse to acquire the Smad2ΔE9,10/+;

Cyp26A1-/- embryos. The original Smad2ΔE9,10/+ female bred to the founder

+/- Cyp26A1 male was of the F4 generation C57BL/6. For breeding involving the

3loxP/3loxP +/- hypomorphic allele, Smad2 F5+3 females were bred to a Cyp26A1 male.

Breeding involving the compound triple heterozygotes were generated by

+/- ΔE9,10/+ +/- breeding a Tgif F5+3 to a Smad2 ; Cyp26A1 animal. Gathered embryos were dehydrated and stored in 100% MeOH at -20oC.

5.4.2 Genotyping

Genotyping of mice was performed by PCR on either tail or toe biopsy,

while genotyping of embryos was on extraembryonic membranes, or embryonic

tissue. For genotyping primers used, see Table 2.2 and Table 4.2. The following

primers sets were used: WT: SM2-16 and R2; Smad23loxP: SM2-16 and R2;

120 Smad2ΔE9,10: SM2-16 and WTR; Tgif-: CTNeo1 and CTNeo2. For alleles specific to this chapter, see Table 5.3. The following primer sets were used: WT: P2 and

P3; Cyp26A1-: P1 and P4. For PCR procedure, see section 2.4.4.

121

Table 5.1 Intercrosses between Smad2/Cyp26A1 and Cyp26A1 result in partially-penetrant HPE. Genotypic analysis of offspring resulting from crosses between mice harboring Smad2/Cyp26A1 and Cyp26A1 mutant alleles and the percentage of embryos displaying HPE. A. Analysis of embryos after E11.5 show that Smad2ΔE9,10/+; Cyp26-/- embryos are lethal at an earlier stage. B. HPE is displayed in 42% of Smad2ΔE9,10/+; Cyp26A1-/- embryos at E9.5-10.5 on a near- congenic C57BL/6 strain background.

122

Table 5.2 Intercrosses between Smad2, Cyp26A1, and Tgif. Genotypic analysis of live born offspring resulting from crosses between mice harboring alleles of Smad2, Cyp26A1, and Tgif mutant alleles. A. Reduction of Smad2 via the use of the Smad23loxP hypomorphic allele does not result in any lethality when combined with Cyp26A1+/-. B. Lethality is observed in compound triple heterozygotes of Smad2, Cyp26A1, and Tgif. All mice are on a near-congenic C57BL/6 background.

123

Table 5.3 Primer sequences.

124

Figure 5.1 Holoprosencephaly phenotype. HPE is observed in a portion of Smad2ΔE9,10/+; Cyp26A1-/- E10.5 embryos on a near-congenic C57BL/6 background. Arrows point to each hemisphere of the forebrain that is present. WT is wild-type.

125

CHAPTER 6

DISCUSSION

6.1 Smad2 Alleles and Phenotypes

TGF-β is a major signaling cascade, being involved in almost every

aspect of development and growth. TGF-β signaling has been observed in

proliferation, apoptosis, differentiation, and gastrulation. The TGF-β superfamily

contains both the TGF-β and BMP signaling cascades, and utilizes the Smad

family of proteins as intracellular mediators. Smad2 and Smad3 are the

intracellular transmitters for TGF-β, nodal, and activin signaling ligands. Upon

ligand binding, the receptors are activated, and in turn activate Smad2 and

Smad3 via phosphorylation of the C-terminal serine residues in the MH2 domain.

These activated mediators then complex with Smad4 and subsequently

translocate into the nucleus. Once in the nucleus this Smad complex, through

interactions with other transcription factors, regulate the expression of target

responsive genes [47, 48, 49, 50].

126 To determine the role and function of Smad2 during mammalian

development, different loss of function alleles have been generated, and each of

these Smad2 alleles result in early embryonic lethality when null [51, 52, 53, 54,

55, 56]. However, these null phenotypes are not all phenotypically identical.

Some phenotypes are empty yolk sacs containing mesoderm, as what is

observed in Smad2Robm1/Robm1 embryos [55], while others are small gastrulation

defects lacking mesoderm and the extraembryonic portion of the egg cylinder, as

what is observed in Smad2ΔC/ΔC embryos [56]. Thus this is different phenotypes

resulting from different Smad2 alleles that disrupted wild-type function via removal of different exons in the genome. It has long been argued that these two dissimilar phenotypes cannot both result from null alleles, and one must be either a hypomorph or an antimorph. This question would be answered with the creation of two new Smad2 alleles, the conditional Smad23loxP allele, which

contains a neo cassette, and the Cre-recombined deletion allele Smad2ΔE9,10

[75].

Previously it has been reported that the neo cassette contains a cryptic splice donor and acceptor site, and insertion of a neo cassette intronically can result in the creation of a hypomorphic allele [66]. Even though homozygous

Smad23loxP mice are obtained at Mendelian ratios and show no observable

abnormality, the neo cassette is aberrantly spliced into some of the Smad2

mRNA transcript. No functional protein is predicted to result from this fusion, so

there would be an overall reduction in wild-type Smad2 protein. Based on real-

time PCR analysis, Smad23loxP/3loxP embryos have a 60.8% reduction of wild-type

127 mRNA. Even though homozygous Smad23loxP embryos have greatly reduced

Smad2 levels, mice are obtained at Mendelian ratios and show no observable

abnormality. Breeding these Smad23loxP mice with a Cre line resulted in the

creation of the recombined allele Smad2ΔE9,10, where portions of the MH2 domain

are removed. Smad2ΔE9,10/ΔE9,10 embryos are indistinguishable from Smad2ΔC/ΔC embryos. Furthermore, we propose that the Smad2ΔE9,10 allele is null for a

variety of reasons. First, exons 9 and 10 of this allele have been removed, and exon 11 is out of the reading frame so will result in an early nonsense codon.

The MH2 domain contains both phosphorylation sites for activation of Smad2 and the L3 loop, which is critical for Smad binding to the Type I receptor to activate Smad2. The Smad2ΔE9,10 allele lacks both these regions, and removal of

the L3 loop results in a non-functional protein [65]. Second, the Smad2ΔE9,10 allele is not antimorphic, as injection of Smad2 mRNA lacking exons 9-11 into

Xenopus embryos did not produce any abnormalities, while a known dominant- negative Smad23SÆA produced anterior truncations [63]. Therefore, we propose

any protein that may be produced from this allele will be non-functional.

Breeding this Smad2ΔE9,10 null allele with the Smad23loxP allele results in lethality at stages later than null embryos, and the Smad2ΔE9,10/3loxP embryos can be

divided into three separate categories. One of the phenotypes observed is the

empty yolk sac, as is observed in the homozygous embryos of the hypomorphic

allele Smad2m1Mag [66] and the Smad2Robm1 allele [55].

128 Considering that there is a difference in the phenotypes observed in

Smad2ΔE9,10/ΔE9,10 and Smad2Robm1/Robm1 embryos, and the Smad2ΔE9,10 allele is a null allele, a possible hypothesis is that the Smad2Robm1 allele is hypomorphic.

Understanding the function of these alleles is vital when trying to elucidate the

role of Smad2 in development or in the progression of diseases and disorders

like cancer. Previously, conflicting results had been reported as to the

progression of intestinal polyps in Smad2/Apc knockout mice, where the only

difference between the two experiments was the use of different Smad2

transgenic alleles. In one assay, there was no effect on progression of the

polyps [147], while in the other there was accelerated progression [148]. Thus, it

is critical to know whether an allele is null, hypomorphic, or antimorphic.

However, there was another difference between the Smad2Robm1 allele

and Smad2ΔE9,10 allele other than the genomic regions that are removed, and that

was strain background. Strain background has a profound effect upon

penetrance and severity of phenotypes. When the Smad23loxP and Smad2ΔE9,10 alleles were bred into the C57BL/6 background from the outbred stock they had been maintained on, a variation in phenotype and penetrance was observed.

Smad23loxP/ΔE9,10 embryos no longer exhibited a variety of phenotypes, but all

displayed an empty yolk sac phenotype. Further, the Smad2ΔE9,10 null embryos

were more variable in the phenotypes that could be exhibited, and displayed both

the gastrulation defect and the empty yolk sac phenotype. The empty yolk sac

phenotype can also be observed in the homozygous Smad2Robm1 embryos.

Thus, there is a possibility that Smad2Robm1, like Smad2ΔE9,10, is a null allele and

129 the resulting phenotype depends on unknown modifier(s) unique to the strain of

mouse used. Alternatively, the Smad2Robm1 allele may be a very severe

hypomorph, but result in a null phenotype nevertheless, so the allele functions as if it was a null. How could two null alleles (Smad2ΔE9,10 and Smad2Robm1) and two

hypomorphic alleles (Smad2m1Mag and Smad23loxP) produce the same

phenotype? Like the null alleles, the hypomorphic alleles (under the conditions

that result in embryonic lethality) fail to attain the threshold requirement of

Smad2/TGF-β signaling required for correct normal development. In

development, being close but under the threshold requirement is no different

than being far from the requirement, the resulting phenotype is still the same

because the minimum threshold requirement was not satisfied. Thus,

Smad2ΔE9,10 is a null allele, Smad23loxP is a hypomorphic allele, the phenotypes

depend upon strain background, and differences in strain background may

explain why various Smad2 null phenotypes are dissimilar.

6.2 Smad2, Retinoid Signaling, and HPE

Mouse and human neural tube development are very similar; thus

studying the mouse will aid in understanding the normal and abnormal process in

humans (reviewed in [6, 7, 8, 9]). Abnormal neural tube development can be due

to genetic or environmental risk factors. Development of the prosencephalon

occurs in three phases, ultimately separating the left and right hemispheres of

130 the telencephalon and the diencephalon. Disturbances early in this patterning

process that disrupt complete cleavage result in some of the most severe neural

tube defects of the CNS, with many cases spontaneously aborting.

Holoprosencephaly is the most common neural tube defect in humans,

affecting 1 out of every 250 conceptions [10]. However, due to the high rate of

lethality associated with this disorder, it is only seen 1 out of every 16, 000 live

births [11]. HPE is a spectrum disorder, encompassing phenotypes of the

prosencephalon ranging from aprosencephlay (an absence of the

prosencephalon) to lobar (nearly complete hemispheric separation). Many

alleles are associated with mammalian cases of HPE as either causative or contributing factors to this disorder [12], and include mutations in SHH [13], TGIF

[14], PATCHED [15], and 7-dehydrocholesterol reductase, which results in

defects in cholesterol biosynthesis that will have an effect on SHH function

(active SHH has a covalent attachment of cholesterol) [149, 150, 151]. However,

in humans, only 70% of carriers of any given mutation display HPE [152].

Additionally, environmental causes have been associated with HPE and include

RA [16], and alcohol [17]. Many human cases have no known cause. Thus, it is

likely that some of these cases of unknown causes are due to mutations in genes

not identified, or mutations in genes that resulted in sensitized embryos that

would have to be exposed to a teratogen at subteratogenic levels, or in

combination with other mutant alleles, before HPE can occur.

131 Previously, it has been reported that reductions in TGF-β signaling components resulted in embryos sensitive to RA teratogenesis [110]. Reduction of Smad2 resulted in embryos that were more sensitive to atRA-induced HPE.

This can be observed with both the null alleles and the hypomorphic alleles.

Using the TGII mouse reporter line, Smad2 heterozygous embryos display variability in retinoid signaling levels. This was more variable than what WAS observed in wild-type littermates, and indicated that misregulation of retinoid signaling WAs occurring. Since retinoid signaling appeared so misregulated, analysis was undertaken to determine which genes were affected that could result in these embryos being sensitive to teratogenic levels of atRA. Two genes involved in the synthesis and degradation of retinoic acid, ADH4 and Cyp26A1, were found to be affected when that analysis was performed on the outbred background. On the C57BL/6 background, only ADH4 continued to be affected.

This decreased ability to survive when challenged with fluctuations in RA observed in Smad2 mutant embryos was also observed in ADH4-/- embryos

[102]. Analyzing the promoter region of ADH4 reveals a number of SBEs clustered in two distinct areas; these are sites where TGF-β signaling can be influencing the expression level of this gene. This would be the first confirmation that TGF-β signaling is influencing and regulating overall retinoid signaling via a

Smad protein. This is by not the only pathway that could be affected, as genes associated with Shh, alcohol metabolism, and cholesterol biosynthesis could also be having an effect upon the normal development and cleavage of prosencephalon.

132 Mutations in TGIF have been associated with human cases of HPE [14,

118, 120]. In mice, TGIF has been identified as a corepressor in both the TGF-β

and retinoid signaling pathways, and reduction of TGIF results in embryos that

have increased retinoid signaling. The Tgif null mouse failed to display HPE on

the outbred background, but compound heterozygotes of Smad2 and Tgif

resulted in a partially-penetrant HPE phenotype (T. Carrel, Ph.D. Dissertation).

Strain background influences the penetrance of phenotypes [127, 128, 129, 130,

131], and strain background would also be important to the penetrance of the

HPE phenotype in Smad2ΔE9,10/+; Tgif +/- embryos. As the Smad2/Tgif mice bred

into our mixed colony, the penetrance of the HPE phenotype these embryos

displayed reduced to 0%; however, breeding near-congenic onto C57BL/6

resulted in a return of the penetrance of the HPE phenotype. Most likely, the reduction of Smad2 sensitized the embryo to the affects of an increase in retinoid signaling that occurred because of reduction of TGIF. This resulted in a portion of the embryos displaying HPE. Decreases in the gene expression levels of both

Cyp26A1 and Cyp26C1 in Smad2ΔE9,10/+; Tgif +/- embryos may contribute to the

observed anterior defects, since anterior CNS defects were observed in

Cyp26A1/Cyp26C1 null embryos, phenotypes reminiscent of teratogenic

exposure to atRA at early stages of development [134]. Decreases in both these

degrading enzymes in Smad2ΔE9,10/+; Tgif +/- embryos could be contributing to an

inability to efficiently remove the atRA, so when the cells are unable to correctly regulate their exposure to atRA, this results in anterior defects including HPE.

Similarly, anterior defects like HPE can be observed in Smad2ΔE9,10/+; Cyp26A1-/-

133 embryos. Complete removal of Cyp26A1, in addition to the affects Smad2

reduction has upon RA metabolism, could result in an environment with

teratogenic amounts of RA. This scenario would be detrimental to the

developing forebrain if teratogenic levels are reached around E8.5-9.5, when the

neural tube is forming and cleavage of the prosencephalon is commencing.

Thus, early affects in retinoid signaling and TGF-β signaling simultaneously can

result in holoprosencephaly, consistent with a multiple-hit theory [131] where

more than one gene needs to be affected before a phenotype is observed.

Considering that the HPE phenotype is a defect in early neural tube patterning, and that early mouse embryos are very sensitive to increases in atRA, the affect of the reduction of Smad2 and TGIF is likely to be the most detrimental at E8.5. At this stage, reduction in expression of Cyp26A1,

Cyp26C1, or ADH4 can be observed in specific combinations of Smad2 and/or

Tgif. In Smad2 mutant embryos ADH4 and Cyp26A1 (depending on strain background) are reduced, thus reducing the ability of these embryos to adequately self-regulate their endogenous retinoid signaling levels. This is, in and of itself, not lethal. However, increased retinoid signaling via exposure to exogenous teratogenic levels of atRA, or increased retinoid signaling via reduction in Tgif, result in a portion of the embryos developing HPE. Removal of all Cyp26A1 and reduction of Smad2 may result in very elevated levels of atRA; as such, nearly all Smad2ΔE9,10/+; Cyp26A1-/- embryos displayed HPE. Tgif

heterozygous embryos displayed a reduction in Cyp26A1 transcription levels,

while Cyp26C1 was reduced in Smad2/Tgif embryos. Reduction in both these

134 RA catabolizing enzymes could be resulting in teratogenic levels of endogenous

RA in the early embryo, resulting in HPE in some of the embryos. Though

retinoid signaling is known to self-regulate, regulate RA metabolism in a

feedback loop, and influence TGF-β signaling, there is now evidence that TGF-β

signaling is affecting retinoid signaling though regulation of RA metabolizing

enzymes (Fig. 6.1).

Similar to human HPE cases, there is not always an absolute direct

correlation between genotype and phenotype. For the model system used in this

analysis, to display the HPE phenotype multiple transgenic alleles had to be

combined to result in HPE in some of the embryos. Thus, Smad2 heterozygous

embryos alone did not result in HPE phenotypes, but did in combination with Tgif

heterozygotes or Cyp26A1 homozygotes, or upon exposure to exogenous RA.

Thus, Smad2 heterozygous embryos were sensitized and when another gene

was affected, or an environmental factor was introduced, this acted in conjunction with the inherent sensitivity unique to each individual embryo, and

periodically resulted in HPE. In the future, TGF-β signaling via Smad2 can be

scrutinized closer as a contributing factor underlying the etiology of human cases

of HPE.

135

Figure 6.1 Model of TGF-β and retinoid signaling pathways. Retinol is converted into retinoic acid via a two-step process. RA then activates retinoid signaling which in turn regulates itself and other gene important for forebrain development like Shh. RA is degraded by catabolizing enzymes. Retinoid signaling can also repress TGF-β signaling. TGIF can repress both retinoid signaling and TGF-β signaling. TGF-β signaling, via Smad2, can influence retinoid signaling through the metabolizing enzymes, and synergistically via TGIF and Smad2 regulate Cyp26C1 expression. Arrows indicate activation, dotted and dashed lines indicate repression, dashed arrow indicates interaction that appears to be strain background dependant. Grey indicates interactions that were already known, while green indicate new interactions based on this work.

136

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