Morphological Pattern and Molecular Signaling During Intervertebral and Epiphyseal Fusion in Cetaceans and Terrestrial Mammals

Home , Georgiacetus, Indocetus, Maiacetus, Vertebral column

MORPHOLOGICAL PATTERN AND MOLECULAR SIGNALING DURING INTERVERTEBRAL AND EPIPHYSEAL FUSION IN CETACEANS AND TERRESTRIAL MAMMALS

A dissertation submitted To Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Meghan Marie Moran

August 2012

Dissertation written by

Meghan Marie Moran

B.A., University of Illinois, Urbana-Champaign, 2002

M.A., Western Michigan University, 2004

Ph.D., Kent State University, 2012

Approved by

______, Chair, Doctoral Dissertation Committee J.G.M. Thewissen ______, Members, Doctoral Dissertation Committee Christopher J. Vinyard ______, Walter E. Horton, Jr. ______, Samuel D. Crish

Accepted by

______, Director, School of Biomedical Sciences Robert V. Dorman ______, Dean, College of Arts and Sciences John R. D. Stalvey ii

TABLE OF CONTENTS

LIST OF FIGURES ...... iv

LIST OF TABLES ...... vii

ACKNOWLEDGEMENTS ...... viii

Chapter Page

I INTRODUCTION ...... 1

II SACRAL ONTOGENETIC ENDOCHONDRAL OSSIFICATION OF THE INTERVERTEBRAL DISC IN MICE (MUS MUSCULUS) ...... 13

III PATTERNS OF INTERVERTEBRAL AND EPIPHYSEAL FUSION IN CETACEANS COMPARED TO TERRESTRIAL MAMMALS ...... 56

IV INTERVERTEBRAL CERVICAL FUSION IN CETACEANS COMPARED TO INTERVERTEBRAL SACRAL FUSION IN TERRESTRIAL MAMMALS ...... 97

V IDENTIFICATION OF THE SACRUM IN MODERN CETACEANS ...... 122

VI A REVIEW OF INTERVERTEBRAL AND EPIPHYSEAL FUSION IN CETACEANS AND TERRESTRIAL MAMMAL ...... 128

REFERENCES ...... 138

iii

LIST OF FIGURES

Figures Page

1 Intervertebral disc diagram ...... 6

2 Regions of the intervertebral disc ...... 14

3 Protein signaling ...... 20

4 Histology of the lumbosacral joint (L6S1) in mouse ...... 32

5 Histology of the first sacral joint (S1S2) in mouse ...... 34

6 Growth plate and blood vessels of intervertebral disc ...... 36

7 Immunohistochemistry of the lumbosacral joint (L6S1) in mouse ...... 38

8 Immunohistochemistry of the lumbosacral joint (L6S1) in mouse ...... 40

9 Immunohistochemistry of the first sacral joint (S1S2) in mouse ...... 42

10 Immunohistochemistry of the first sacral joint (S1S2) in mouse ...... 44

11 Protein expression similar in non-fusing and fusing intervertebral joints ...... 46

12 protein expression different between non-fusing and fusing intervertebral joints ...... 48

13 VonKossa histology for mineralization in mouse ...... 51

14 Land mammal sacra ...... 68

15 Archaic cetacean sacra...... 70

16 Bowhead whale cervical specimens and cervical fusion maps ...... 72

17 Mouse postnatal ontogenetic fusion maps ...... 74

LIST OF FIGURES (Continued)

Figure Page

18 Pig postnatal ontogenetic fusion maps ...... 76

19 Archaic cetacean fusion maps ...... 77

20 Bowhead and beluga whale fusion maps...... 79

21 Four articulation points altered by evolution mapped on an alpaca pelvis ...... 81

22 Cetacean phylogeny of sacral fusion ...... 82

23 Cleared and stained Stenella attenuata ...... 87

24 Phylogeny of cervical fusion related to neck length ...... 90

25 Semicircular canal size overlaid on the cervical fusion and neck length data ...... 93

26 Intervertebral disc diagram ...... 98

27 MMP13 protein expression in bowhead cervical intervertebral discs ...... 105

28 TIE2 protein expression in bowhead cervical intervertebral discs ...... 107

29 GDF5 protein expression in bowhead cervical intervertebral discs ...... 108

30 BMP2/4 protein expression in bowhead cervical intervertebral discs ...... 110

31 Skeletonized bowhead whale ontogenetic cervical series ...... 111

32 CT scans of bowhead neck ...... 113

LIST OF FIGURES CONTINUED

Figure Page

33 Bowhead body length compared to neck length ...... 114

34 Cleared and stained bowhead fetus ...... 114

35 Intervertebral fusion patterns of bowhead whale and mouse ...... 115

36 Mouse and bowhead immunohistochemistry results ...... 124

37 Pudendal nerve of modern cetaceans ...... 125

38 Cleared and stained cetacean fetuses ...... 132

LIST OF TABLES

Table Page

1 Bowhead Whale (Balaena mysticetus) Samples ...... 102

2 Mouse and Bowhead Immunohistochemistry Results...... 117

vii

ACKNOWLEDGEMENTS

First I would like to thank my advisor, Dr. Hans Thewissen for his unwavering support and encouragement during my progress as a graduate student and during the completion of this dissertation. Hans provides unique research opportunities, excellent discussion, and a genuine attitude towards his students and science. I am truly thankful to him for the wonderful opportunities he has provided for me.

I thank Dr. Walt Horton for accepting me into his lab in my first year of graduate school and providing me with an excellent environment to learn lab techniques and engage in scientific discovery. Dr. Chris Vinyard is thanked for his excellent advice, instruction, and discussions on science. Dr. Sam Crish is thanked for serving on my committee and his support during this dissertation process. Thank you to Dr. Mary Ann Raghanti for acting as Graduate Student

Representative and Moderator during my defense.

Others I would like to extend a thank you to are, Dr. Dean Dluzen of

NEOMED who helped me start my first mouse colony; Dr. Bill Lynch and his graduate students Jaclyn Stenger and Sandra Cardona also of NEOMED for donating mouse cadavers; Dr. Makarand Risbud and his technician Renata

Skubutyte of Thomas Jefferson University for their completion of ANK immunohistochemistry and collaboration; Dr. Terrance Demos of Loyola

viii

University Medical Center for his willingness to donate his and his departments services in CT scanning fossil and modern whale vertebrae; Dr. John Mort and

Dr. Eunice Lee of Shriners Hospital Laboratories, McGill University, Montreal,

Canada for their generous donation of Collagen X antibody; Jason Herreman of the North Slope Borough Department of Wildlife Management for his hard work and heavy lifting during Balaena mysticetus sample collection; Denise McBurney of NEOMED for all her scientific support, friendship, and laughs; Sharon Usip of

NEOMED for her patience, guidance, and teaching in the lab; Beth Lowder for troubleshooting Von Kossa staining; and Lee Brucato, my last summer student, who worked on mouse immunohistochemistry.

I received excellent administration and support from Diane Kehner,

Debbie Heeter, Diana Dillon, Debbie Severt, Margaret Weakland, and Judy

Wearden. I would like to thank the library staff especially Lisa Barker, Laura

Colwell, and Denise Cardon for all their hard work in collecting sources during this project and their willingness to search for those hard to find pieces of literature. I also thank the Comparative Medicine Unit staff, Lora Nicholson,

Debbie Dutton, Linda McCort, and Dr. Walter Horne for their support and guidance with my mouse colony and IACUC protocols.

I would like to thank Dr. J. Craig George and Dr. Robert Suydam of the

Department of Wildlife Management in Barrow, AK; the Alaska Eskimo Whaling

Commission, and local subsistence hunters; Dr.Terrence Demos and Dr. John F.

Moran of Loyola University Medical Center for their roles in completing the CT scans of the whales; Dr. Sirpa Nummela of the University of Helsinki for her role in this project; Dr. Larry Heaney and John Mead of the Field Museum of Natural

History and Dr. Sue McLaren of the Carnegie Museum of Natural History for their help with museum collections; Dr. Emily Buchholtz for her advice.

There are many fellow (and former) graduate students who supported me throughout my graduate career and through the process of this dissertation.

Without their discussions, guidance, and support, this project would not be possible: Dr. Lisa Noelle Cooper, Dr. Tobin L. Hieronymus, Dr. Ashleigh Nugent,

Dr. Tremie Gregory, Dr. Brooke A. Armfield, Dr. Amy Mork, and Alison Doherty.

Lastly, I would like to thank Mike and our families; John, Cabrina,

Stephanie, Chris, Laura, Joseph, Lorraine, Jolene, and Steve, as well as my friend Kate for their unwavering support, excitement for my progress, and love that kept me going throughout my graduate career.

I truly thank you all!

CHAPTER I

INTRODUCTION

Mammalian skeletons are diverse in bony morphology and exhibit morphologies specific to functions and environmental factors. Two morphological features of the vertebral column are intervertebral fusion and epiphyseal fusion; these two types of fusion are the focus of this dissertation.

Changes in skeletal morphology, specifically intervertebral and epiphyseal fusion, are clearly visible in cetaceans (whales, porpoises, and dolphins) as skeletal adaptations. The vertebral column of cetaceans is the subject of a number of studies (Slijper, 1936, Wheeler, 1930; Slijper, 1936; Stewart and Stewart, 1989;

Haldiman and Tarpley, 1993; Buchholtz, 2001; Buchholtz et al., 2005; Buchholtz,

2007; Buchholtz, 2010). In this study, I approached cetacean vertebral morphology in two modern cetacean species (Balaena mysticetus,

Delphinapterus leucas), with specific attention to the lack of fusion between sacral vertebrae, intervertebral fusion in the cervical vertebrae, and delayed epiphyseal fusion in vertebrae. I interpret these morphological patterns against a backdrop of comparative anatomy of laboratory mammals (Mus musculus) as well as non-model species (Sus scrofa). These fusion patterns are also investigated in archaic cetacean taxa. I pursued morphological, paleontological,

1 2 molecular, and developmental study of the intervertebral disc and epiphyses of the vertebrae.

The theme of this research is variation. Variation is a substrate for evolution to act upon. Variation has implications for natural selection and evolution, specifically, in this case, cetacean evolution and the morphological changes that occur in the vertebral column in the land to water transition. I aim to further understanding of mammalian morphological and molecular variation and expand scientific understanding of the relationship between phenotype and genotype (Willmore et al., 2007). Variation in phenotype and genotype directly affects natural selection and therefore has major implications for development

(Willmore et al., 2007). These processes are cyclic and continually affect one another (Willmore et al., 2007). These morphological changes in phenotype and genotype are most likely based on the alteration of gene expression It is this gene expression that I aim to understand during intervertebral fusion in the sacrum of mice and necks of bowhead whales and determine if these mechanisms behind intervertebral fusion are similar or variable across mammals.

Early Development of the Vertebral Column

In the early embryo there are three types of tissue or cell lineages- mesoderm, endoderm, and ectoderm. The mesoderm adjacent to the neural tube is called paraxial mesoderm and will give rise to somites; the notochord is derived from the mesoderm as well (Hirsinger et al., 2005; Gridley 2006; Gilbert,

2010). The paired somites are condensations of sclerotic cells (O’Rahilly and

Meyer, 1979; Fan and Tessier-Lavigne, 1994), which will give rise to vertebrae and intervertebral discs (Christ and Wilting, 1995). In humans, there are 42-44 somitic pairs, 50 somites in chicks, 65 somites in mice, and in bowheads the exact number is unknown; but all somites are not present at the same time

(O’Rahilly and Meyer, 1979). The number of somites increases ontogenetically and as each vertebra is formed the somites disappear. The number of somites does not correlate to the number of vertebrae present in the fully developed animal (Richardson et al., 1998).

During development of the vertebral column the sclerotome of the somites is resegmented (Christ and Wilting, 1992; Gilbert, 2010). Resegmentation is the process in which each vertebra is formed by the cranial half of one somite and the caudal half of preceding somite (Christ and Wilting, 1992; Gilbert, 2010). The mesenchymal sclerotome forms the vertebrae and intervertebral discs of the vertebral column as well as the axial muscles (Christ and Wilting 1992; Hirsinger et al., 2005; Gridley 2006).

Joint Formation with Special Attention to Intervertebral Joints

To provide background on joint morphology, there are three categories of joints: 1) syntharthroses are immovable joints, 2) amphiarthroses are joints with limited range of movement, and 3) synovial joints are totally mobile (Archer et al.,

2003). Most joint development and formation research usually focuses on synovial joints (Später et al., 2006). Joints between the articular surfaces of the

4 vertebrae are synovial, but the joints between the centra are amphiarthroses, joints where bony elements are separated by a cartilaginous disc and allow for minimal movement (Winslow and Burke, 2010). Amphiarthroses are designed for stability and to absorb force (Vertechy and Parenti-Castelli, 2006). With limited movement in each intervertebral joint, the range of motion is increased when multiple amphiarthroses are stacked together as in the vertebral column

(Vertechy and Parenti-Castelli, 2006).

Synovial joint formation is understood in more detail than formation of other joint types and is summarized here because I will use it as a reference at times during this work. During synovial joint formation the joint interzone is the location where a joint will form (Später et al., 2006). Cells in the interzone are dense and flattened losing the characteristics of early chondrocytes (Hartmann and Tabin 2001; Später et al., 2006). These flat cells in the joint interzone produce types I and III collagens (Später et al., 2006). The interzone cells down- regulate extracellular matrix (ECM) components, which contributes to forming a gap between skeletal elements (Hartmann and Tabin, 2001). The interzone can be divided into three layers; each layer is comprised of decreasing cell density

(Hartmann and Tabin, 2001). The central intermediate lamina is the central layer where cells undergo apoptosis leading to joint cavitation (Hartmann and Tabin,

2001). Joint cavitation is the process by which a space is formed between developing skeletal elements (Archer et al., 2003). This is the potential joint

5 space and is formed when cells undergo apoptosis and the extracellular matrix is degraded by matrix metalloproteinases (Archer et al., 2003).

Intervertebral Disc Formation and Intervertebral Disc Regions

There are three main regions of the intervertebral disc (Fig. 1). These regions are the annulus fibrosus, endplates, and nucleus pulposus (Walker and

Anderson, 2004). Densely packed mesenchymal cells gives rise to the annulus fibrosus (Chris and Wilting 1992) the outermost region of the intervertebral disc.

Both parts of the annulus fibrosus are composed of highly organized type I collagen fibers (Antoniou et al., 1996; Roughley, 2004; Walkers et al., 2004;

Shankar et al., 2009; Risbud et al., 2010; Hristova et al., 2011; Smith et al., 2011) and fibroblastic cells (Dahia et al., 2009). The annulus fibrosus attaches to the endplates (Coventry et al., 1945; Taylor, 1975; Dahia et al., 2009), which are two regions of hyaline cartilage located at the cranial and caudal ends of each intervertebral disc, adjacent to the vertebral bodies (Coventry et al., 1945;

Roberts et al., 1989, Moore, 2006; Shankar et al., 2009; Smith et al., 2011). The endplates are characterized by large, round, hypertrophic chondrocytes (Dahia et al., 2009). The function of the endplates is to provide nutrients to the intervertebral disc (Hristova et al., 2011), prevent the nucleus pulposus from being extruded from the disc (Moore, 2000; Melrose et al., 2008), and provide structural support to the intervertebral joint (Roberts et al., 1989; Moore, 2000).

Between the endplates, the nucleus pulposus is located in the center of the

vertebra vertebra

Horizontal section of a generalized intervertebral disc with all regions labeled.

Figure 1. Intervertebral disc diagram

7 intervertebral disc. The nucleus pulposus is surrounded by the annulus fibrosus and the former is primarily composed of type II collagen and proteoglycans

(Oegema, 1993; Antoniou et al., 1996; Dahia et al., 2009; Hristova et al., 2011).

Cells of the nucleus pulposus are different from those found in the annulus fibrosus and the endplates; they are derived from notochordal cells and referred to as nucleus pulposus cells (Trout et al. 1982; Christ and Wilting, 1992;

Roughley, 2004; Risbud et al., 2010). Ontogenetically, the number of notochordal cells decreases and chondrocyte-like cells infiltrate the nucleus pulposus (Risbud et al., 2010). Together these four regions are responsible for shock absorption (Coventry et al., 1945), resistance to compression, and mobility of the mammalian spine (Oegema, 1993). In this dissertation, the outer and inner annulus fibrosus tend to be discussed together unless unique protein expression or morphology is exhibited.

The intervertebral disc is in close proximity to the growth plate and epiphysis of the vertebra, but is a separate entity. Growth plates are located at the cranial and caudal ends of each vertebra and are responsible for longitudinal vertebral growth. Vertebral growth plates in humans tend to cease bone growth and close around twenty years of age (Coventry et al., 1945b). Together with intervertebral discs and vertebrae, the growth plate and epiphyses account for vertebral column length.

Endplate Terminology Controversy

Endplates, also called cartilage plates, were thought to be part of the vertebral body by Coventry et al., (1945a). They refer to the endplates as part of the vertebral body because the endplates are perforated by multiple holes or canals allowing blood vessels to pass. Coventry et al., (1945a) distinguish the endplates into three regions, the central zone, peripheral zone, and epiphyseal ring; differentiation of the central and peripheral zones is based on the number of canals present. The terminology is confusing in Coventry et al., (1945a) because the endplate is referred to once as cartilage and later the epiphyseal ring is referred to as bone. The epiphyseal ring surrounds the outside of the endplate

(Coventry et al., 1945a). From further description, it seemed the epiphyseal ring is another term for the bony epiphysis of the vertebra (Coventry et al. 1945b).

Based on the descriptions of the fibers of the annulus fibrosus inserting over the edges of the epiphyseal ring, Coventry et al., (1945a, b) the epiphyseal ring is the mineralized endplate. Endplates are considered part of the intervertebral disc

(Taylor, 1975; Roberts et al., 1989; Moore, 2006; Shankar et al., 2009; Smith et al., 2011) and are considered to be a major structural component of the intervertebral disc that supports the survival of the intervertebral disc itself.

Intervertebral Disc Degeneration and Fusion

Intervertebral discs are large, avascular, hypoxic structures that undergo ontogenetic compositional changes leading to disc degeneration (Ariga et al.,

2001; Risbud and Shapiro, 2001). If any region of the intervertebral disc is damaged a cascade of degeneration is started. The number of healthy cells decreases as senescent cell number in the intervertebral disc increases during disc degeneration (Kim et al., 2009). Disc degeneration is partly due to reduction in nutrient diffusion (Urban et al., 1977; Andersson, 1993; Buckwalter, 1995;

Ariga et al., 2001; Bibby and Urban, 2004; Smith et al., 2011), age (Lauerman et al., 1992; Buckwalter, 1995; Gruber and Hanley, 1998; Ariga et al., 2011), and loading history (Kurowski and Kubo, 1986; Adams et al., 2000; Adams and

Roughley, 2006). Degeneration can start in the nucleus pulposus with cell numbers decreasing and extracellular matrix alteration (Walker and Anderson,

2004). Degeneration progresses to the annulus fibrosus as it loses the lamellar arrangement which alters mechanical properties of the disc (Walker and

Anderson, 2004).

Some intervertebral discs are preprogrammed to degenerate and apoptose (Ariga et al., 2011; Zhao et al., 2006; and Bertram et al., 2009). These discs are located in the sacrum of terrestrial mammals (Grassé, 1967; Simoens et al., 1983; Evans, 1993; Ocal et al., 2006; Belcastro et al., 2008; Rios et al.,

2008; Saber 2008) and the necks of some cetacean taxa (Wheeler, 1930; Slijper,

1936; Stewart and Stewart, 1989; Haldiman and Tarpley, 1993; Buchholtz, 2001;

Buchholtz et al., 2005; Buchholtz, 2007; Buchholtz, 2010).

Molecules of Interest

Seven molecules were chosen including matrix metalloproteinase 13

(MMP13), tyrosine kinases that contain immunoglobulin-like loops and epidermal growth factor- similar domains (TIE2 or TEK), progressive ankylosis (ANK), growth differentiation factors 5 and 6 (GDF5 and GDF6), type X collagen

(Col10α1), and bone morphogenetic proteins 2 and 4 (BMP2/4). These were chosen for their roles in endochondral ossification specifically during extracellular matrix degradation, angiogenesis and blood vessel remodeling, joint maintenance, and bone formation.

Epiphyseal Fusion and Endochondral Ossification

Epiphyseal fusion occurs when the growth plate, a highly organized region of cartilage responsible for longitudinal growth, is fully resorbed (Abad et al.,

2002; Kronenberg, 2003; Mackie et al., 2008; Emons et al., 2011; Mackie et al.,

2011). The epiphysis is a secondary center of ossification, located on the opposite side of the growth plate. The epiphysis attaches the diaphysis (shaft) of a long bone or the centra of a vertebra (Abad et al., 2002; Emons et al., 2011) and growth ceases. The growth plate is comprised of six zones of chondrocytes

(Hunizker and Schenk, 1989; Kronenberg, 2003; Mackie et al., 2008; Mackie et al., 2011). These six zones, from the epiphysis, are resting zone, proliferative zone, columnar zone, prehypertrophic zone, hypertrophic zone, and the calcified cartilage zone (Reno et al., 2006; Mackie et al., 2011). Chondrocytes differentiate through each growth plate zone similar to moving on a treadmill; this

11 chondrocyte differentiation and ultimately chondrocyte death drives bone growth

(Hunziker and Schenk, 1989; Kronenberg, 2003; Reno et al., 2006; Mackie et al.,

2011). This zonal differentiation is tightly controlled by a signaling pathway, the parathyroid hormone related peptide and Indian hedgehog (PTHrP-Ihh) feedback loop (Mackie et al., 2008; Hojo et al., 2010). This feedback loop controls the progression of bone growth from the growth plate (Mackie et al., 2008; Hojo et al., 2010).

The process of epiphyseal fusion is considered part of endochondral ossification. Endochondral ossification is one of two bone formation process; the other is intramembranous ossification. During endochondral ossification a cartilage model, like that of the growth plate, is replaced with bone tissue

(Kronenberg, 2003; Nakashima and de Crombrugghe 2003; Mackie et al. 2008;

Hojo et al., 2010; Mackie et al., 2011). Endochondral ossification forms the majority of the skeleton except for the craniofacial flat bones or lateral portion of the clavicle (Boyan et al., 1990; Mackie et al., 2008; Mackie et al., 2011). There are molecular markers associated with endochondral ossification; I have chosen to study the protein expression a selection of these molecules in the intervertebral disc during intervertebral fusion.

Hypothesis: Genes expressed during endochondral ossification are expressed during intervertebral fusion.

Aims:

Specific aims pursued in this dissertation:

1) To determine if endochondral ossification of long bones is a model for

intervertebral fusion, specific molecules of interest were analyzed

including MMP13, TIE2, COLX, and BMP2/4). Other molecules studied

include ANK, GDF5, and GDF6.

2) To identify the ontogenetic patterns of intervertebral and epiphyseal fusion

within the vertebral column of terrestrial and aquatic mammals to further

understand mammalian variation in an evolutionary context.

3) To determine the ontogenetic intervertebral fusion patterns in the cervical

intervertebral discs of bowhead whales (Balaena mysticetus)

4) To determine if the patterns of protein expression exhibited in sacrum of

terrestrial mammals (mouse) are the same as in the neck of some modern

cetaceans (bowhead whale) in order to identify.

CHAPTER II

ONTOGENETIC ENDOCHONDRAL OSSIFICATION OF THE SACRAL INTERVERTEBRAL DISC IN MICE, (MUS MUSCULUS)

Introduction

To understand intervertebral fusion and expand current fusion treatment techniques, I investigated intervertebral fusion of the sacrum in a mouse model

(Mus musculus). The molecular mechanisms of intervertebral fusion are poorly understood and elucidating the molecular changes that occur in the intervertebral disc during normal intervertebral fusion can help expand the clinical application of spine fusion surgery techniques.

The intervertebral disc is comprised of three cartilaginous regions; the annulus fibrosis, endplate, and nucleus pulposus (Fig. 2). The annulus fibrosus is the outermost layer of the intervertebral disc. It is composed of tough, concentric lamellae of type I collagen-rich fibrocartilage (Antoniou et al., 1996;

Roughley, 2004; Shankar et al., 2009; Risbud et al., 2010; Hristova et al., 2011;

Smith et al., 2011) and fibroblastic cells (Dahia et al., 2009). The annulus fibrosus attaches to the endplates (Coventry et al., 1945; Dahia et al., 2009), which are two regions of hyaline cartilage located at the cranial and caudal ends of each intervertebral disc (Fig. 2), adjacent to the vertebral bodies (Coventry et al., 1945; Roberts et al., 1989, Moore, 2006; Shankar et al., 2009; Smith et al., 13 14

A- horizontal section of the S1S2 intervertebral disc of a 20 day old mouse (Mus musculus). B- black and white line drawing of the horizontal section in A. Regions of the disc are labeled for orientation; af- annulus fibrosus, ep- endplate, gp- growth plate, np- nucleus pulposus, S1- first sacral vertebra, S2- second sacral vertebra.

Figure 2. Regions of the intervertebral disc

2011). The endplates (Fig. 2) are characterized by hypertrophic chondrocytes

(Dahai et al., 2009). The function of the endplates is to provide nutrients to the intervertebral disc (Hristova et al., 2011), prevent the nucleus pulposus from extrusion from the disc (Moore, 2000; Melrose et al., 2008), and provide structural support to the intervertebral joint (Roberts et al., 1989; Moore, 2000).

Between the endplates, the nucleus pulposus is located in the center of the intervertebral disc (Fig. 2). The nucleus pulposus is surrounded by the annulus fibrosus and the former is primarily composed of type II collagen and proteoglycans (Oegema, 1993; Antoniou et al., 1996; Dahia et al., 2009; Hristova et al., 2011). Cells of the nucleus pulposus are different from those found in the annulus fibrosus and the endplates; they are derived from notochordal cells and referred to as nucleus pulposus cells (Trout et al. 1982; Roughley, 2004; Risbud et al., 2010). Ontogenetically, the number of notochordal cells decreases over time allowing chondrocyte-like cells to infiltrate the nucleus pulposus (Risbud et al., 2010). Together these three regions are responsible for shock absorbsion

(Coventry et al., 1945), resistance to compression, and mobility of the mammalian spine (Oegema, 1993). When one or more of these intervertebral disc regions becomes degenerated, surgical intervention is sometimes required, specifically artificial spinal fusions, to alleviate back pain symptoms.

Spinal intervertebral fusion surgeries are performed in the United States

200,000 times a year (Luther et al., 2011). These surgeries have a failure rate of

10-40% as a result of nonunion and failure of healing (Luther et al., 2011). Most of these fusion surgeries occur in the cervical or lumbar region of the human vertebral column (Boden et al., 1995); under normal conditions these joints never fuse (Williams et al., 1995).

One common reason for spine fusion surgeries is to reduce pain associated with intervertebral disc degeneration (Smith et al., 2011). The cartilaginous intervertebral disc undergoes ontogenetic compositional change contributing to disc degeneration (Ariga et al., 2001), partly due to reduction in nutrient diffusion (Urban et al., 1977; Andersson, 1993; Ariga et al., 2001; Bibby and Urban, 2004; Smith et al., 2011). The intervertebral disc is a large avascular structure and in order to maintain cell survival, nutrient diffusion needs to occur efficiently through the endplates and the annulus fibrosus (Brodin, 1955; Urban et al., 1977; Holm et al., 1981; Melrose et al., 2008). Specifically, blood vessels in the endplates are the source of nutrients (Brodin, 1955; Holm et al., 1981; Bibby and Urban, 2004; Roughley, 2004), which are absorbed by extracellular matrix of the intervertebral disc (Holm et al., 1981; Bibby and Urban, 2004). The nutrient diffusion is a gradient; therefore cells closest to the endplate blood vessels will receive more nutrients compared to the nucleus pulposus cells in the middle of the disc (Bibby and Urban, 2004). Nutrient diffusion decreases as the endplates mineralize with increasing age (Roberts et al., 1989; Roughley, 2004; Gruber et al., 2008). Eventually, the nutrient supply becomes too low to support the

17 chondrocytes and cartilaginous extracellular matrix, and the disc chondrocytes apoptose (Ariga et al., 2001). Chondrocyte apoptosis potentially provides an opportunity for bone formation on the extracellular matrix scaffold within the intervertebral disc by means of endochondral ossification.

Endochondral ossification is one of the two bone formation processes.

During endochondral ossification, a cartilage model is replaced with a deposition of calcium and phosphate (Kronenberg, 2003; Nakashima and de Crombrugghe

2003; Mackie et al., 2008; Hojo et al., 2010; Mackie et al., 2011). Endochondral ossification forms the majority of the skeleton except the craniofacial flat bones and lateral portion of the clavicle (Boyan et al., 1990; Mackie et al., 2008; Mackie et al., 2011). I hypothesize that some genes expressed in endochondral ossification are also involved in intervertebral fusion. I tested specific molecules characteristic of endochondral ossification: MMP13 for extracellular matrix degradation (Reboul et al., 1996; Stickens et al., 2004; Monfort et al., 2006),

TIE2 for angiogenic qualities and blood vessel remodeling (Abdulmalek et al.,

2001), type X collagen expressed by terminally differentiated chondrocytes during endochondral ossification (Kwan et al., 1997; Gress and Jacenko, 2000;

Hojo et al., 2010; Stoyanov et al., 2011), and BMP2/4 for osteogenic and ectopic bone formation qualities (Rosen, 2006; Rosen, 2009). Other molecules I pursued but are not associated with endochondral ossification were ANK as

18 mineralization inhibition (Skubutyte et al., 2010) and GDF5 and GDF6 for their joint maintenance qualities (Hötten et al., 1996).

In most mammals, the sacrum is the only region of the vertebral column where multiple vertebrae fuse together to form a solid bony structure (Williams et al., 1995). I aim to understand the molecular changes occurring in normal, non- pathological intervertebral fusion in the mammalian sacrum of a mouse. I followed the histological and biochemical changes within the sacral intervertebral disc during ontogeny of the mouse and compared ontogenetic protein signaling in the non-fusing lumbosacral joint (L6S1) to the first fusing sacral joint (S1S2). I expected MMP13 and TIE2 to decrease during ontogeny because MMP13 will degrade the extracellular matrix to allow for blood vessel infiltration and TIE2 will support the progression of vascularization of the disc; type X collagen and

BMP2/4 will increase ontogenetically as bone formation increases in the intervertebral disc. I expected GDF5 and GDF6 protein expression to decrease ontogenetically during intervertebral fusion. These molecules have multiple functions, and our predictions are based on their roles during joint loss in the vertebral column. I also expect protein expression of the mineralization inhibitor,

ANK, to decrease in the intervertebral disc as mineralization occurs in the intervertebral disc.

To pursue the hypothesis, I utilized histology and immunohistochemistry to evaluate ontogenetic changes in protein expression of the molecules of interests.

I divided endochondral ossification into stages, loosely based on those outlined in Mwale et al., (2000), that are integral during intervertebral disc extracellular degeneration and sacral formation.

Intervertebral Disc Cartilage Degeneration

Occurring first during intervertebral disc degeneration and sacral formation is cartilage extracellular matrix degradation. Extracellular matrix components such as aggrecan and collagens are cleaved by matrix metalloproteinases

(MMPs) (D’Angelo et al., 2000; Cawston and Young, 2010). Some specific matrix metalloproteinases are responsible for extracellular matrix degradation exclusively during endochondral ossification (D’Angelo et al., 2000; Hayami et al., 2000; Uusitalo et al., 2000; Ortega et al., 2004). One such endochondral ossification matrix metalloproteinase is MMP13, also called Collagenase 3

(D’Angelo et al., 2000; Hayami et al., 2000; Uusitalo et al., 2000; Ortega, et al.,

2004; Strickens et al., 2004). MMP13 is a true collagenolytic matrix metalloproteinase with some gelatinolytic properties (Knäuper et al., 1996;

Reboul et al., 1996; Strickens et al., 2004; Monfort et al., 2006). MMP13 is expressed in hypertrophic chondrocytes (Fig. 3) and its signaling is important in the breakdown of collagen types I and II fibers as well as aggrecan in the extracellular matrix (Mitchell et al., 1996; Reboul et al., 1996; D’Angelo et al.,

2000; Hayami et al., 2000; Uusitalo et al., 2000; Ortega et al., 2004; Strickens et al., 2004; Monfort et al., 2006; Lee et al., 2009; Cawston and Young, 2010; Hojo

The protein signaling for the proteins of interest- MMP13, TIE2, ColX, ANK, GDF5, GDF6, and BMP2/4 characterized in a generic cell.

Figure 3. Protein signaling

21 et al., 2010; Mackie et al., 2011). The breakdown of collagen type II is significant in intervertebral fusion because it is a major component of the annulus fibrosus

(Risbud et al., 2010). The cleavage of collagen fibers, specifically collagen type

II, is the rate-limiting step in cartilage breakdown during endochondral ossification (Zijlstra et al., 2004). Extracellular matrix degradation by MMP13 occurs just prior to angiogenesis (Zijlstra et al., 2004; Hojo et al., 2010). MMP13 also promotes vascular invasion (Pogue and Lyons, 2006) suggesting that

MMP13 is critical to endochondral ossification (Stickens et al., 2004; Pogue and

Lyons, 2006). MMP13 is expressed in pathologically degenerated discs

(Shankar et al., 2009), but we predict MMP13 protein expression to be present at the beginning of normal disc degeneration in the sacrum, before mineralization begins.

Angiogenesis and Blood Vessel Infiltration

Terminally differentiated hypertrophic chondrocytes are thought to be mostly responsible for vascular invasion of cartilage (Hojo et al., 2010). The formation of blood vessels, angiogenesis, is a necessary step in endochondral ossification (Deckers et al., 2002) because cartilage is an avascular tissue, and bone requires blood supply. Blood vessels need to infiltrate the degraded cartilage extracellular matrix of the intervertebral disc to ensure survival of newly forming bone tissue. A distinct set of signaling pathways are involved at different stages of angiogenesis (Patil et al., 2012). One such receptor is TIE2 (Tek), an

22 endothelial marker and receptor tyrosin kinase (Fig. 3) (Wong et al., 1997;

Yancopoulos et al., 2000; Eklund and Olsen, 2006). TIE2 is a cell surface receptor that binds to Angiopoetin 1 (Ang1) and Angiopoetin 2 (Ang2)

(Abdulmalek et al., 2001). Ang1 activates TIE2 and this pathway is responsible for blood vessel maturation (Abdulmalek et al., 2001), capillary remodeling during angiogenesis (Wong et al., 1997; Yancopoulos et al., 2000; Eklund and Olsen,

2006), and stabilization of already formed vasculature (Abdulmalek et al., 2001).

TIE2 is stimulated and expressed in highly hypoxic environments (Wong et al.,

1997; Makinde and Agrawal, 2008), therefore the intervertebral disc is an ideal environment (Risbud et al., 2006; Hiyama et al., 2011). The function of TIE2 signaling is understudied in adult tissues (Wong et al., 1997) and particularly in the intervertebral disc. We predict TIE2 expression to occur simultaneously or just after MMP13 expression because TIE2 is responsible for blood vessel remodeling in the changing environment of the intervertebral disc during endochondral ossification.

Bone Formation

The last stage of endochondral ossification in the sacral intervertebral disc is hypertrophy of chondrocytes and bone formation (Kronenberg, 2003; Mackie et al., 2008). Hypertrophic chondrocytes are terminally differentiated cells and this maturation induces a change in extracellular matrix component synthesis

(Mackie et al., 2011). Changes in hypertrophic chondrocyte synthesis include

23 the decrease of type II collagen and the initiation of type X collagen (Mwale et al.,

2000; Kronenberg, 2003; van der Eerden et al., 2003; Mackie et al., 2011). Type

X collagen is secreted and localized extracellularly adjacent to hypertrophic chondrocytes (Fig. 3).

Type X collagen is a hypertrophic chondrocyte marker specific to endochondral ossification (Wallis et al., 1996; Hristova et al., 2011; Mackie et al.,

2011). Some functions of type X collagen include supporting bone formation during degradation of cartilage matrix, facilitating removal of type II collagen fibers, participating in mineralization, and influencing vascular invasion of the cartilage extracellular matrix (Rosati et al., 1994). Hypertrophic chondrocytes eventually undergo apoptosis and the residual extracellular matrix acts as a scaffold for osteoblasts and bone formation (Kronenberg, 2003; Mackie et al.,

2008).

Bone formation during endochondral ossification starts with the formation of hydroxyapatite crystals in the extracellular matrix scaffold (Kronenberg, 2003;

Mackie et al., 2008; Mackie et al., 2011). These crystals form adjacent to hypertrophic chondrocytes, which express specific bone morphogenetic proteins

(BMPs). BMPs were first identified for their ability to form bone ectopically (Urist and Strates, 1971) and have been shown to be crucial for endochondral ossification (Rosen, 2009). BMP2 and BMP4 are two closely related bone morphogenetic proteins that have osteogenetic qualities (Duprez et al., 1994;

Sato et al., 1999; Ducy and Karsenty, 2000Chen et al., 2004; Wan and Cao,

2005) and share 86% of their amino acid sequence (Wozney et al., 1990). BMP2 is expressed by hypertrophic chondrocytes (Wan and Cao, 2005; Pogue and

Lyons, 2006). I studied the expression of type X collagen in hypertrophic chondrocytes and BMP2 and BMP4 during bone infiltration of the intervertebral disc during sacral intervertebral formation. I predicted type X collagen and

BMP2/4 protein expression increases during sacral formation in the intervertebral disc.

Inhibition of Mineralization

Once the cartilaginous disc extracellular matrix is degraded sufficiently for blood vessel infiltration and remodeling, mineralization and osteoblast invasion of the extracellular matrix occurs (Fig. 3). Mineralization and bone formation are strictly regulated processes (Aszódi et al., 2000; Hall, 2005; Martin and Sims,

2006) regardless of location within the skeleton. There are many genes responsible for controlling the process of mineralization. One such gene is ANK; it is a multi-pass transmembrane protein that regulates inorganic pyrophosphate

(PPi) transport into and out of the cell (Ho et al., 2000; Ryan, 2000; Terkeltaub,

2001; Sohn et al., 2002; Johnson et al., 2003; Zaka et al., 2009; Skubutyte et al.,

2010). PPi inhibits calcification and mineralization by inhibiting the formation of hydroxyapatite crystals, and thus ANK controls mineralization (Ho et al., 2000;

Johnson et al., 2003; Wang et al., 2005; Skubutyte et al., 2010). When PPi is

25 hydrolyzed extracellularly it becomes inorganic phosphate (Pi), which contributes to hydroxyapatite crystal formation. Pi stimulates chondrocyte terminal differentiation and apoptosis leading to mineralization (Kim et al., 2010). The

ANK signaling pathways is dependent upon the amount of oxygen available in tissues (Skubutyte et al., 2010). Specifically, ANK is expressed in low levels in hypoxic conditions and higher in regions with increased oxygen (Zaka et al.,

2009). Therefore, we predict that ANK expression is low in the nucleus pulposus of the disc because the nucleus pulposus is extremely hypoxic, while ANK expression should be increased closer to the endplates, where there are higher levels of oxygen. Also, we predict that ANK expression will be decreased during intervertebral fusion because the intervertebral joint is no longer being maintained (Gurley et al., 2005), therefore ANK expression would no longer be necessary to inhibit mineralization of the intervertebral disc.

Joint Loss and Bone Formation

GDF5 and GDF6 (growth differentiation factors 5, 6) are members of the

BMP family of proteins (Settle et al., 2003; Sena et al., 2007) and therefore members of the TGF-β superfamily (Settle et al., 2003; Coleman et al., 2011).

GDF5 and GDF6 bind to BMP1 receptors and BMP2 receptors on the cell surface (Fig. 3), signaling through the Smad pathway into the nucleus to regulate transcription of downstream GDF targets (von Bubnoff and Cho, 2001).

During development, GDF5 and GDF6 are expressed in a striped pattern across the developing potential joint space in both the appendicular skeleton

(Storm and Kingsley, 1996; Wolfman et al., 1997; Francis-West et al., 1999;

Buxton et al., 2001; Settle et al., 2003) and vertebral synovial joints (Settle et al.,

2003). In mouse embryos at E14.0, GDF5 is expressed in the annulus fibrosus of the mouse spine, but is absent from the nucleus pulposus and the developing vertebral bodies (Mundy et al., 2011). GDF5 has been shown to be essential for intervertebral disc differentiation and maintenance (Dahia et al., 2009).

GDFs share 40-50% of their protein sequence with BMP2 and BMP4

(Spiro et al., 2001) and also share BMP receptors, suggesting that sequence homology may support the ability to GDFs to form ectopic bone (Hötten et al.,

1996; Erlacher et al., 1998). Studies have shown that GDF5 induces spinal fusions in adult mice (Coleman et al., 2011), has roles in skeletogenesis (Hötten et al., 1996; Francis-West et al., 1999; Coleman et al., 2011) and the regulation of bone repair (Coleman et al., 2011), and induces angiogenesis. All of these roles suggest that GDF5 is integral in supporting bone growth (Yamashita et al.,

1997; Sena et al., 2007; Coleman et al., 2011). Due to the lack of research on the role of GDFs in joint maintenance of postnatal and adult joint tissue, I expect

GDF5 and GDF6 protein expression is decreased during intervertebral fusion.

Anatomical Abbreviations

C, cervical vertebra; T, thoracic vertebra; L, lumbar vertebra; S, sacral vertebra; and Ca, caudal vertebra are used to define vertebral regions within the vertebral column. Each abbreviation is followed by a number indicating the location of the vertebra within each region. Lower numbers are cranial within the vertebral region.

Methods and Materials

Tissue Preparation

A postnatal ontogenetic series of wild-type CD-1 mice (Mus musculus) consisting of both sexes were collected for eight time points, every 10 days from

Day 1 (birth) - Day 60. Mice 447 days old were also collected. All mice were fed and watered ad libitum and housed in a 12 hour light/dark cycle room. Animals under 10 days postnatal were euthanized using an abdominal injection of Fatal

Plus. Animals older than 10 days postnatal were CO2-euthanized according to

Northeast Ohio Medical University Institutional Animal Care and Use Committee

(NEOMED IACUC) protocol 09-020.

All sacral joints (S1-S4) were assessed for dorsoventral histological and protein expression differences within the intervertebral disc to determine if sacral intervertebral fusion is uniform across the disc. No differences were identified and therefore I focused on the median of the disc in a horizontal plane. Also, the

28 three sacral joints in the mouse were evaluated for ontogenetic intervertebral disc changes during fusion. From this analysis, the S2S3 and S3S4 joints demonstrate the same protein expression pattern, but the timing of this expression is delayed in the last two sacral joints of the mouse. Therefore, only descriptions of the S1S2 protein expression will be presented here.

The vertebral samples used for immunohistochemistry and Safranin-O histology were harvested and fixed in 10% buffered formalin for 1-2 weeks depending on the age of the animal. Vertebral samples were then decalcified in

10% ethylenediaminetetraacetic acid (EDTA) for 2-4 weeks. After decalcification, the vertebral columns were washed in 1XPBS and the L6-S4 joints were excised.

Each of the vertebral samples were dehydrated in graded ethanol and xylene washes and embedded in Fisherbrand Paraplast X-tra paraffin. All vertebral samples were cut at six micron thick horizontal sections using a Leica RM 2165 microtome. Sections were mounted on Superfrost Plus microscope slides

(Fischer Scientific).

Undecalcified postnatal mouse vertebral samples were collected for

VonKossa mineral stain (Bills et al., 1971). Vertebral columns were harvested and fixed using 10% buffered formalin for seven days. After fixation, the vertebral columns were washed in 1XPBS and the L6-S4 vertebral region was prepared for processing. Each of these samples were dehydrated in graded ethanol and xylene washes and embedded in Richard Allen Type H paraffin and

29 cut on a Leica RM 2165 microtome at 3 µm. Sections were mounted on

Superfrost Plus microscope slides (Fischer Scientific). The entire thickness of each vertebral sample was cut and the entire joint was reviewed as to any dorsoventral change in protein signaling.

Histology

Safranin-O histology was completed on the ontogenetic mouse series to visualize the cartilage bone interface, intervertebral disc morphology, and proteoglycan content, which changes with age and amount of fusion. Safranin-O attaches to the proteoglycans in cartilage and is identified by bright red staining

(Rosenberg, 1971; Camplejohn and Allard, 1988). Slides were deparaffinized and rehydrated in graded ethanol and xylene washes. The slides were soaked in

Weigert’s hemotoxylin for 6 minutes, followed by hydrochloride and ammonium hydroxide washes. Slides were then immersed in 0.1% fast green for 1 minute and 0.1% Safranin-O for 6 minutes. Slides were dehydrated and coverslipped using DPX paramount.

VonKossa mineral staining was completed to identify calcium phosphate content in the lumbosacral and sacral intervertebral joints. The timing and pattern of mineralization in the three sacral joints in mice were examined. All glassware used for VonKossa staining was cleaned thoroughly with a 50% bleach solution and autoclaved. Undecalcified paraffin sections were deparaffinized and rehydrated as outlined above. Slides were submerged in 1%

30 silver nitrate under a 250 watt incandescent bulb for 1 hour followed by 3 minutes in 2.5% sodium thiosulfate, and 6 minutes in 0.1% Safranin-O solution. Every

10th slide of the dorsoventral width of the centrum was stained with VonKossa to evaluate mineralization ontogenetically. Slides were dehydrated and coverslipped using DPX paramount.

Immunohistochemistry

MMP13, TIE2, GDF5, GDF6, and BMP2/4 expression in the intervertebral disc was assessed using immunohistochemistry. After deparaffinization and rehydration in graded ethanol and xylene washes, antigen retrieval was completed using 85oC sodium citrate (pH=7) for 10 minutes. Primary antibody concentrations for GDF5, GDF6, MMP13, and TIE2 were 1:500; BMP2/4 antibody concentration was 1:250 (Santa Cruz Biotechnology Inc). All primary antibodies were incubated at 4oC overnight. Localization of the primary antibody was completed with the ABC anti-goat (sc-2023) or ABC anti-rabbit (sc-2018) staining kit (Santa Cruz Biotechnology) following manufacturer’s protocol. Slides were counterstained using 0.01% thionin and coverslipped using DPX mounting media. All experiments included a control slide for which primary antibody was omitted.

The type X collagen antibody was a generous gift from Dr. Eunice Lee and

Dr. John Mort of Shriner’s Hospital in Montreal, CA. After deparaffinization, rehydration in ethanol, and xylene washes, antigen unmasking was completed

31 using 1% bovine testis hyaluronidase (Sigma) in a hydration chamber at 37oC for

45 minutes. Endogenous peroxidase activity was quenched using 1% H2O2 solution. Localization of primary antibody with a 1:50 concentration was accomplished using anti-rabbit (sc-2018) staining kit (Santa Cruz) following manufacturer’s protocol. Slides were counterstained using .01% thionin and coverslipped using DPX paramount. All experiments included a control slide for which primary antibody was omitted.

Slides for the immunolocalization of ANK (from the lab of MR) were humidified with a TBS solution, deparaffinized, and rehydrated in a series of graded ethanol and xylene washes. Vector antigen unmasking solution was used for antigen retrieval and a hydrogen peroxide solution was used to quench endogenous peroxidase activity. Slides were incubated overnight with the primary antibody at 1:100 concentration at 4oC. Slides were then washed in a series of TNT solutions and antibody detection was completed in a dark room.

Histology Results

The lumbosacral joint (L6S1) is just cranial to the sacrum and does not fuse under normal developmental conditions in mice (Fig. 4). The lumbosacral intervertebral disc endplates begin to mineralize by 10 days postnatal (Fig. 4A).

No other mineralization is detected in the lumbosacral disc. The annulus fibrosus of the L6S1 intervertebral disc decreases in size as ontogeny proceeds as shown in VonKossa and Safranin-O histology (Fig.4). The annulus fibrosus maintains a

VonKossa and Safranin-O histology. A- 10 days, B- 20 days, C- 30 days, D- 40 days, E- 50 days, F, 100 days (VonKossa) and over one year (Safranin-O). Notice the increase in mineralization from A to B in the endplate and the ontogenetic change in composition of the nucleus pulposus. All sections are midline in the horizontal plane. 10x.

Figure 4. Histology of the lumbosacral joint (L6S1) in mouse

33 highly fibrous appearance with few chondrocytes present (Fig. 4). The composition of the L6S1 nucleus pulposus changes ontogenetically: from 30 days postnatal, the nucleus pulposus is highly cellular with a thin ring of extracellular matrix present along the outside of the nucleus pulposus (Fig. 4A-

C), while by 40 days postnatal, the nucleus pulposus becomes dominated by extracellular matrix (Fig. 4D), which continues through one year postnatal (Fig.

4E and F) and the red Safranin-O stain for proteoglycans increases in the nucleus pulposus ontogenetically (Fig. 4).

The S1S2 endplate of the intervertebral disc begins to mineralize by 10 days postnatal (Fig. 5A) like in the L6S1 joint and by 20 days the outer annulus fibrosus is mineralized as well (Fig. 5B). This pattern of mineralization continues as development proceeds until the intervertebral disc is completely mineralized with the exception of the nucleus pulposus (Fig. 5A-C). The nucleus pulposus is maintained in the first sacral intervertebral disc past 60 days postnatal (Fig. 5F), but the periphery of the intervertebral disc is mineralized completely by 30 days postnatal (Fig. 5C). The mobility of the S1S2 joint is limited due to this peripheral mineralization and therefore considered fused at 30 days postnatal. The nucleus pulposus of the S1S2 intervertebral disc decreases in size ontogenetically (Fig.

5). Proteoglycan concentration is low or completely lacking in the annulus fibrosus of the S1S2 intervertebral disc as shown by the lack of Safranin-O staining in the disc tissue (Fig. 5).

VonKossa and Safranin-O histology. A- 10 days (10x), B- 20 days (10x), C- 30 days (10x), D- 40 days (10x and 40x), E- 50 days (10x and 40x), F- > one year (10x and 40x). Notice the mineralization of the S1S2 intervertebral disc in the VonKossa images. The intervertebral disc is completely infiltrated by bone by one year of age (F). All sections are midline in the horizontal plane.

Figure 5. Histology of the first sacral joint (S1S2) in mouse

Growth Plate Histology

All zones of the long bone growth plate are present and identifiable in the vertebral growth plate at 10 days postnatal (Fig. 6A and B). The width of the growth plate decreases with increasing age (Fig. 5) due to a decrease in the number of cells and decreased cell activity. The extracellular matrix of the growth plate is rich in proteoglycans as indicated by the deep red Safranin-O staining (Figs. 4-6). The staining of the growth plate cartilage is different from the staining present in the intervertebral disc; there is much less Safranin-O staining and therefore less proteoglycan content in the disc when compared to the growth plate.

Blood Vessel Infiltration

By 20 days postnatal blood vessels are present in the annulus fibrosus and the endplates of the S1S2 joint (Fig. 6C and D). Blood vessels are present evenly across the dorsoventral gradient of the intervertebral disc. The blood vessels start as small, endothelial structures with red blood cells in their lumen.

The invasion of the intervertebral disc by blood vessels continues through 60 days postnatal (Fig. 6) expanding into all regions of the intervertebral disc except the nucleus pulposus. By one year postnatal, the intervertebral disc is completely remodeled and bone is present in all regions of the intervertebral disc

(Fig. 6F). Osteocytes are visible in the intervertebral joint and look the same as the bone of the vertebral body (Fig. 6F).

Safranin-O. A- 30 day old mouse S1S2, 10x; B- close up of growth plate of A, 40x; C- 20 day old mouse S1S2 joint; D- 60 day old mouse S1S2 joint. Notice the growth plate zones in A and B. Notice the blood vessels forming in the intervertebral disc in C and D (black arrows). Growth plate zones labeled: cc- calcified cartilage zone, h- hypertrophy zone, c- columnar zone, r- resting zone. Horizontal sections.

Figure 6. Growth plate and blood vessels of the intervertebral disc

L6S1 Immunohistochemistry Results

MMP13 signaling in the intervertebral disc decreases ontogenetically from 10 to

40 days postnatal, with heavy nuclear staining in the annulus fibrosus and endplate chondrocytes (Fig. 7A). The cell membranes and the nuclei of the nucleus pulposus cells also exhibit MMP13 protein expression. By 60 days postnatal, MMP13 protein expression is decreased in all regions of the intervertebral disc (Fig. 7A) and by over one year old (447 days postnatal),

MMP13 signaling is absent.

Through 30 days postnatal, the L6S1 intervertebral disc exhibits TIE2 protein expression in the cytoplasm of annulus fibrosus and endplate chondrocytes (Fig. 7B). At 50 days postnatal TIE2 protein expression decreases in the cytoplasm of endplate and annulus fibrosus chondrocytes and this decrease continues through 60 days postnatal at which time TIE2 is absent from the intervertebral disc (Fig. 7B).

Type X collagen (Col10α1) protein expression is present in the extracellular matrix of the endplate and inner annulus fibrosus of 10-60 days postnatal L6S1 intervertebral joint. Type X collagen protein expression is absent in the nucleus pulposus throughout the ontogenetic series (Fig. 7C).

In a 20 day postnatal mouse, BMP2/4 protein expression is present in the cytoplasm of endplate and inner annulus fibrosus chondrocytes as well as nucleus pulposus cells (Fig. 7D). This pattern of BMP2/4 expression is

A- MMP13, B- TIE2, C- ANK, in 20, 40, and 60 day postnatal. Notice the decrease in protein expression in the intervertebral disc as age increases. The insert provided for reference on location within the intervertebral disc (red square). Labels: af- annulus fibrosus, ep- endplate, gp- growth plate, np- nucleus pulposus, v- vertebra. Horizontal sections, 40x.

Figure 7. Immunohistochemistry of the lumbosacral joint (L6S1) in mouse

39 maintained through 60 days postnatal though there is a slight decrease (Fig. 7D).

Once mice are over a year old, BMP2/4 protein expression is greatly reduced to isolated endplate chondrocytes and nucleus pulposus cells.

Heavy nuclear, cytoplasmic, and cell membrane ANK protein expression is present in endplate and annulus fibrosus chondrocytes from 20 to 60 days postnatal (Fig. 8A). The nuclei and cell membranes of nucleus pulposus cells are also positive for ANK protein expression. There is a slight decrease in ANK protein expression in all regions of the intervertebral disc at 60 days postnatal, but ANK expression remains present.

GDF5 protein expression is present in the cytoplasm of endplate chondrocytes and nucleus pulposus cells at 10 and 20 days postnatal (Fig. 8B).

By 30 days postnatal, GDF5 signaling is present in the annulus fibrosus as well as the endplate and nucleus pulposus. At 40 days postnatal, GDF5 signaling is decreased slightly in the annulus fibrosus and endplate chondrocytes and by 60 days postnatal, GDF5 signaling is decreasing in all regions of the intervertebral disc, but remains present. This decrease in expression continues through 1 year of age until GDF5 is absent. The nucleus pulposus cells, however, maintain

GDF5 signaling past 60 days postnatal.

GDF6 protein expression is similar to that of GDF5 in the L6S1 intervertebral joint, except that protein expression is maintained into one year postnatal in the endplate and nucleus pulposus (Fig. 8C).

A- GDF5, B- GDF6, C-COLX, D- BMP2/4, in 20, 40, and 60 day postnatal. The insert provided for reference on location within the intervertebral disc (red square). Labels: af- annulus fibrosus, ep- endplate, gp- growth plate, np- nucleus pulposus, v- vertebra. Horizontal sections, 40x.

Figure 8. Immunohistochemistry of the lumbosacral joint (L6S1) in mouse

Sacral Joint IHC Results

MMP13 protein expression is present in the nucleus of annulus fibrosus and endplate chondrocytes and in the nuclei and cell membranes of nucleus pulposus cells (Fig. 9A) through 40 days. By 60 days, MMP13 expression decreases (Fig. 9A) and by one year, MMP13 expression is absent in the S1S2 intervertebral disc.

Through 30 days postnatal TIE2 expression is present in the cytoplasm of annulus fibrosus and endplate chondrocytes and in the cell membranes of the nucleus pulposus cells (Fig. 9B). Decrease of TIE2 protein expression begins at

30 days postnatal and continues through 50 days postnatal in the annulus fibrosus and the endplate, expression remains present in the cell membranes of nucleus pulposus cells. TIE2 signaling is absent by 60 days in the intervertebral disc (Fig. 9B).

Type X collagen (Col102α) protein expression is maintained in the intervertebral disc extracellular matrix from 10 days through one year of age.

There is no type X collagen in the nucleus pulposus at any time point (Fig. 9C).

BMP2/4 protein expression is present in the cytoplasm of annulus fibrosus and endplate chondrocytes as well as the cell membranes of nucleus pulposus cells (Fig. 9D). BMP2/4 protein expression starts to decrease at 50 days postnatal in all regions of the intervertebral disc and continues to decrease through one year of age.

Figure 9. Immunohistochemistry of the first sacral joint (S1S2) in mouse

The S1S2 joint exhibits the same ANK protein expression pattern from 20 through 60 days postnatal (Fig. 10A) with expression present in the cell membrane, cytoplasm, and nuclei of annulus fibrosus and endplate chondrocytes as well as the cell membranes, cytoplasm, and nuclei of nucleus pulposus cells.

GDF5 and GDF6 exhibit the same protein expression in the S1S2 joint and therefore are discussed together. GDF5/6 protein expression is present in the cytoplasm of endplate chondrocytes and nucleus pulposus cells in the intervertebral disc at 10 days postnatal. By 20 days, GDF5/6 protein expression increases in the annulus fibrosus chondrocytes and is maintained in the endplate chondrocytes and nucleus pulposus cells (Fig. 10B and C) through 40 days at which time GDF5/6 protein expression starts to decrease and by 50 days postnatal GDF5 expression is decreased in the annulus fibrosus; the outer annulus fibrosus and endplate chondrocytes still exhibit strong GDF5 expression intracellularly as in the nucleus pulposus cells. At 60 days postnatal GDF5 expression is decreased significantly in all regions of the disc except the nucleus pulposus, which maintains GDF5 and GDF6 expression through one year of age

(Fig. 10B and C).

Discussion

This study begins to fill a void in intervertebral disc research by focusing on protein expression ontogenetic changes in the sacral intervertebral disc of mice. The sacrum is a vertebral region that normally undergoes intervertebral

A- GDF5, B-GDF6, C-COLX, D- BMP2/4, in 20, 40, and 60 day postnatal. Notice the decrease in protein expression in the intervertebral disc as age increases. Horizontal sections, The insert provided for reference on location within the intervertebral disc (red square). Labels: af- annulus fibrosus, ep- endplate, gp- growth plate, np- nucleus pulposus, v- vertebra. Horizontal sections, 40x40x.

Figure 10. Immunohistochemistry of the first sacral joint (S1S2) in mouse

45 fusion. I tested whether endochondral ossification is a model for sacral fusion and formation because the intervertebral disc is cartilage that undergoes ontogenetic infiltration by bone. Some differences were identified in the ontogenetic patterns of protein expression examined in this paper between a non-fusing (L6S1) and a fusing (S1S2) intervertebral joint.

Protein Expression in Non-Fusing and Fusing Intervertebral Joints

I found that protein expression within a non-fusing and fusing intervertebral disc is more similar than expected (Fig. 11). MMP13, TIE2, GDF5, and type X collagen yielded very similar protein expression patterns in the non- fusing and fusion joints (Fig. 11). These results were surprising because, for example, MMP13 was expected to degrade cartilage extracellular matrix in the

S1S2 fusing joint to allow for TIE2 and angiogenic infiltration. This was not the case for sacral intervertebral disc. The overall protein expression patterns agree with our predictions that MMP13 and TIE2 protein expression should decrease ontogenetically. GDF5 protein expression was predicted to be the same as

GDF6, which it was not. GDF5 expression decreased though this decrease was the same in both the L6S1 and the S1S2 joints. Type X collagen protein expression does not support our prediction because type X collagen protein expression remains constant ontogenetically and there was no change in expression between the non-fusing and fusing joints.

MMP13, TIE2, GDF5, and COLX protein expression are similar between non-fusing (L6S1) and fusing (S1S2) intervertebral joints.

Figure 11. Protein expression similar in non-fusing and fusing joints

ANK, GDF6, and BMP2/4 protein expression results were different in the non-fusing L6S1 and in the fusing S1S2 intervertebral joints (Fig. 12). ANK protein expression is maintained in the L6S1 joint through 60 days postnatal at which time protein expression decreases slightly (Fig. 12). This does follow my prediction for ANK expression and therefore the hypothesis is rejected; ANK is a mineralization inhibitor and was expected to decrease as the integrity of the cartilaginous intervertebral disc was lost ontogenetically and during fusion, but the decrease in expression was more delayed than expected and does not correspond to the onset of fusion.

GDF6 protein expression was maintained to one year postnatal in the

L6S1 nucleus pulposus and endplate; expression decreased in the annulus fibrosus around 60 days postnatal (Fig. 12). In the S1S2 joint, GDF6 decreases protein expression around 60 days in all regions of the intervertebral disc. There are differences in GDF6 protein expression present between a non-fusing and fusing joint; results suggest GDF6 is maintained in the nucleus pulposus and endplates for a longer amount of time in the L6S1 non-fusing joint when compared to the S1S2 fusing joint.

BMP2/4 protein expression is different in the L6S1 and S1S2, but results do not support my hypothesis (Fig. 12). BMP2/4 protein expression is maintained through 60 days in L6S1, but in the fusing S1S2 joint expression is shorter and decreases around 50 days postnatal. For an osteogenetic growth

ANK, GDF6, and BMP2/4 protein expression patterns are slightly different between non- fusing (L6S1) and fusing (S1S2) intervertebral joints.

Figure 12. Protein expression different between non-fusing and fusing intervertebral joints

49 factor, we expected more intense expression and a longer duration of expression in the fusing S1S2 joint where bone is forming in the disc.

The histology and immunohistochemistry of all sacral joints (S1 through

S4) yielded the same staining and protein expression patterns, but the timing of expression is delayed in the S2S3 and S3S4 sacral joints compared to the first sacral joint (S1S2). The first sacral joint (S1S2) starts fusing at 20 days postnatal in mice and the S2S3 joint starts fusing later at about 30 days postnatal. The

S3S4 sacral joint exhibits mineralized endplates and little more intervertebral disc mineralization past 100 days postnatal.

Epiphyseal Fusion Controversy in Rodents

It has been stated that rodents never fuse their epiphyses (Donaldson,

1924; Dawson, 1925; Kennedy et al., 1999; Weise et al., 2001; van der Eerden et al., 2002; Emons et al., 2011), though it was later shown that rodents (e.g. mice, rats, guinea pigs) do close their growth plates, but fusion is delayed (Dawson,

1925; van der Eerden 2002). Rodents do not fuse growth plates at the end of puberty like other mammals (Gilsanz et al., 1988; Emons et al., 2011). Instead rodent growth plates remain open into adulthood (Kennedy et al., 1999).

Dawson (1925) showed 1270 day old rats exhibit resorbed growth plates; no cartilage is present. In this paper, we confirm that growth plates in mice are retained past one year of life, but the cell activity and amount of growth after this time is unknown.

The Endplate: Disc or Vertebra

Endplates, also called cartilage plates were thought to be part of the intervertebral disc by Coventry et al., (1945). These authors refer to the endplates as part of the vertebral body because these structures are perforated by holes or canals allowing blood vessel passage. Coventry et al., (1945) divides the endplates into three regions, the central zone, peripheral zone, and epiphyseal ring; differentiation of these three zones is based on number of canals present. The terminology is confusing in Coventry et al., (1945) because the endplate is referred to once as cartilage and later the epiphyseal ring is referred to as bone. By the 1950’s it was decided the endplates were part of the intervertebral disc (Taylor, 1975; Roberts et al., 1989; Moore, 2006; Shankar et al., 2009; Smith et al., 2011). The endplates are considered to be a major structural component of the intervertebral disc that supports the survival of the intervertebral disc itself.

Endplate Mineralization

It has been shown that the endplates become mineralized in the lumbar region of the vertebral column and therefore nutrient diffusion becomes difficult

(Gruber et al., 2005), but the lumbar intervertebral discs remain intact. We confirm that regardless of vertebral region, all endplates mineralized (Fig. 13).

There are differences in non-fusing and fusing intervertebral joints that go beyond mineralization of the endplates. There must be some other molecular

Cervical, lumbar, and sacral horizontal sections, 10x. Notice the endplate is mineralized in all vertebral regions starting before 20 days postnatal.

Figure 13. VonKossa histology for mineralization in mouse

52 mechanisms preventing the cervical and lumbar intervertebral discs from degenerating too early and fusing all vertebral levels.

The sacrum of humans fuses completely by about 30 years (Broome et al., 1998; Belcastro et al., 2008) and in mice by 30 days postnatal (Figs. 5 and

13). Disc degeneration has been documented in humans as early as late teens and early 20’s (Shankar et al., 2009) in lumbar spine. Though degeneration occurs relatively early in life, this level of degeneration is maintained for a long time. To understand other mechanisms at work in the intervertebral discs, more research is needed.

Clinical Application

This study can be applied clinically in regard to expanding surgical spine fusion techniques. Techniques used today to fuse vertebrae together include those utilized for nonunion fractures (Angle et al., 2012), autografts (Boden and

Schimandle, 1995; Olabisi et al., 2011), allografts (Boden and Schimandle,

1995), and molecularly soaked scaffolds (Boden et al., 1995; Olabisi et al.,

2011). Of these techniques, the gold standard for spine fusion is the autograft, where bone is harvested from the iliac crest and implanted in the vertebral region to be fused (Boden and Schimandle, 1995). The autograft promotes osteogenesis and support to the fusing vertebral joint (Boden and Schimandle,

1995). This technique though has high infection rate in the implant site and the donor site (Boden and Schimandle, 1995; Olabisi et al., 2011). This same

53 process can be use an allograft or bone from a cadaver or donor. Progress with spine fusion surgical techniques expanded to molecularly soaked scaffolds.

Molecularly soaked scaffolds are sponges made from a variety of media including coral (Damien et al., 1993) and collagen (Schimandle et al., 1995). The scaffolds are soaked in a variety of osteoinductive molecules including BMP2

(Schimandle et al., 1995; Olabisi et al., 2011), BMP6, BMP7, BMP9, and GDF5.

The osteoinductive success of these molecules is dependent upon the dose and the timing of the release (Boden et al., 1995).

Conclusion

I was able to identify the pattern of sacral intervertebral disc fusion in a mouse model. Though in mice the periphery of the sacral intervertebral joint is mineralized by 30 days postnatal, the nucleus pulposus is maintained past 60 days postnatal. I consider the sacral joint fused by 30 days due to the amount of mineralization and limitation in mobility of the joint. From VonKossa mineralization histology I confirm all endplates mineralize regardless of fusion status for vertebral level as shown in Bernick et al. (1991). If all endplates mineralize regardless of vertebral level and therefore limit nutritional diffusion to all intervertebral discs, the question arises why do the majority of intervertebral disc remain cartilaginous? There must be other molecular mechanisms controlling intervertebral disc degeneration and mineralization in sacral discs, or

54 inhibitory factors present in all non-fusing intervertebral joints that are responsible for maintaining cartilaginous intervertebral discs.

At this time I am unable to determine if endochondral ossification is the cause of intervertebral disc ossification and therefore intervertebral fusion. I suggest there may be two different and independent mechanisms occurring in the disc. Endochondral ossification may occur in the annulus fibrosus and the endplates, but not in the nucleus pulposus, which is maintained past 60 days postnatal and retains protein expression of some joint maintenance molecules

(GDF5 and GDF6). This joint maintenance occurring in the nucleus pulposus may be associated with the difference in cell type and lineage between the annulus fibrosus and the nucleus pulosus, but this is not confirmed in this study and further research is needed.

Protein expression in the intervertebral discs of the mouse sacrum yielded more similarities between non-fusing and fusing joints than expected. Three of the seven proteins (ANK, GDF6, and BMP2/4) yielded different expression patterns between the non-fusing and fusing joints. MMP13, TIE2, GDF5, and type X collagen protein expression was very similar between non-fusing and fusing joints ontogenetically. Again, this supports the expansion of proteins of interest characteristic of endochondral ossification to determine if endochondral ossification is the bone formation process occurring in the intervertebral disc during intervertebral fusion. There may be two processes occurring in the

55 intervertebral disc depending on the intervertebral disc region. By understanding the molecular mechanisms of intervertebral fusion in a normal, non-pathological model, such as mouse sacrum, artificial means of fusion can be expanded for surgical application in humans.

CHAPTER III

PATTERNS OF INTERVERTEBRAL AND EPIPHYSEAL FUSION IN CETACEANS AND TERRESTRIAL MAMMALS

Introduction

The hallmark characteristic of all vertebrates is the segmented vertebral column (Gegenbaur and Bell, 1878; Hyman, 1922), which is comprised of multiple vertebrae and intervertebral discs (Gegenbaur and Bell, 1878; Hyman,

1922; Williams et al., 1995). Alternating bony vertebrae and cartilaginous intervertebral discs provide the torso with flexibility and mobility in life (Gilbert,

2010; Hristova et al., 2011; Bruggeman et al., 2012). Ontogenetic and evolutionary changes occurring within the vertebral column impact locomotor style and life history of vertebrates, for instance, by rendering aquatic mammals incapable of terrestrial locomotion.

The vertebral column consists, in all vertebrates, of a series of alternating bony vertebrae and cartilaginous intervertebral discs provides the torso with flexibility and mobility in life and this combination provides the body with support, flexibility, and mobility (Gilbert, 2010; Hristova et al., 2011; Bruggeman et al.,

2012). Given the great diversity of body forms and locomotor modes within vertebrates, it is to be expected that the shape and number of vertebrae and

56 57 intervertebral discs varies significantly. In terrestrial mammals the vertebral column connects to the limbs and supports much of the weight of the animal

(Williams et al., 1995, Romer, 1958). However, the cetacean (whales, porpoises, and dolphins) vertebral column does not have a function in counteracting gravity; its locomotor function is concentrated in the distal part of the tail, where the fluke is located.

My interest in the vertebral column of cetaceans was instigated by the extensive morphological observations by Slijper (1936) and Buchholtz (1998;

2001; 2007; 2010; Buchholtz) who exposed many of the differences between cetaceans and terrestrial mammals. The focus of this study is on the pattern of ossification in the intervertebral discs, which, inevitably, reduces flexibility and mobility. As part of my interest in variation in skeletal morphology, I study ossification in the vertebral column of cetaceans against the comparative background of some terrestrial mammals. There are three morphological factors

I specifically and interested in during cetacean evolution: reduction of the hind limb, the loss of sacral fusion, and the loss of pelvic articulation of the sacrum.

These morphological changes are very different from the morphology exhibited in terrestrial mammals. The identification and comparison of vertebral fusion patterns in extant and extinct mammalian taxa are pursued using dissection, whole mount clearing and staining, osteological analysis, and computerized tomography (CT). The timing of intervertebral and epiphyseal fusion patterns will

58 also be explored ontogenetically and evolutionary in some terrestrial mammals and cetaceans.

Ontogenetic and evolutionary changes occurring within the vertebral column impact locomotor style and life history of vertebrates by rendering derived aquatic mammals incapable of aquatic locomotion. Vertebral column morphology and morphological changes occurring within the vertebral column contribute to diversity among mammals. Vertebral morphology and mammalian diversity can be traced evolutionarily and ontogenetically to understand how extant mammalian morphology was derived.

One important morphological feature in the vertebral column is bony fusion or synostosis. The fusion of vertebrae provides stability and strength to the vertebral column (Boden & Schimandle, 1995), a structure that contains multiple joints (Gegenbaur & Bell, 1878; Hyman, 1922; Williams et al., 1995).

Stability and strength of the axial skeleton are important factors for efficient locomotion (Gingerich et al., 1990; Mulder, 2001; Buchholtz, 2007; Adam, 2009).

Intervertebral fusion occurs between adjacent vertebral bodies or centra as well as the zygapophyses, laminae, and pedicles of the vertebra. Here, we focus on vertebral centra synostoses because it is an important during sacral formation. The fusion of vertebral centra provides stability and strength to the sacrum (Boden & Schimandle, 1995) and this structural strength helps supports terrestrial locomotion. Sacral intervertebral fusion begins when the intervertebral disc degenerates and undergoes apoptosis (Ariga et al., 2001; Zhao, Jiang &

Dai, 2006; Bertram et al., 2009) resulting in fusion of the centra in the sacrum

(Evans, 1993; Williams et al., 1995; Bab et al., 2007). The number of synostosed sacral vertebrae and the rate of sacral synostosis varies across Mammalia

(Grassé, 1967; Simoens et al., 1983; Evans, 1993; Ocal, Ortanca & Parin, 2006;

Belcastro et al., 2008; Rios, Weisensee & Rissech, 2008; Saber 2008).

Unusual patterns of intervertebral fusion occur in some cetaceans and sirenians. In these aquatic mammals, sacral vertebrae never fuse as intervertebral discs remain cartilaginous and do not undergo apoptosis.

However, intervertebral fusion does occur in some cetaceans in the cervical vertebrae (Wheeler, 1930; Slijper, 1936; Stewart & Stewart, 1989; Haldiman &

Tarpley, 1993; Buchholtz et al., 2005; Buchholtz, 2010). The number of fused cervical vertebrae varies among cetacean species and ontogeny (Wheeler, 1930;

Slijper, 1936; Stewart & Stewart, 1989; Haldiman & Tarpley, 1993; Buchholtz,

2001; Buchholtz et al., 2005; Buchholtz, 2007; Buchholtz, 2010).

Epiphyseal fusion occurs when the cranial and caudal vertebral epiphyses, or secondary ossification centers, fuse to the vertebral centrum during ontogeny (Young, 1957; Williams et al., 1995; Currey, 2002). In juvenile mammals, the epiphyses are separated from the centrum by a cartilaginous growth plate; this is referred to as an open growth plate. While the growth plate is open or unfused, longitudinal bone growth can occur (Kronenberg, 2003;

Mackie et al., 2008; Mackie et al., 2011). Growth plates are also found in developing long bones, and eventually these epiphyses ossify with the diaphysis

60 or vertebral centrum and bone growth ceases (Ballock & O’Keefe, 2003; Nilsson

& Baron, 2004; Lui et al., 2010; Emons et al., 2011; Mackie et al., 2011). The function of the growth plate and epiphyseal fusion is the same across mammalian taxa regardless of location in the skeleton (Wheeler, 1930; McKern and Stewart, 1957; Ohsumi, Nishiwaki & Hibiya, 1958; Silberberg, 1971; Kato,

1988; Moran & O’Connor, 1994; Best & Lockyer, 2002; Martin, Ritman & Turner,

2003; Cardoso, 2008; Munro, Weinstock, 2009; Albert et al., 2010).

Sacral Intervertebral Fusion

The multiple vertebra sacrum of terrestrial mammals was reduced to a single vertebra and eventually lost pelvic articulation during the Eocene in the land to water transition to extant cetaceans (Thewissen et al., 2001; Gingerich,

2003; Bajpai, Thewissen & Sahini, 2009; Thewissen et al., 2009). The reduction of the hind limb, the loss of sacral fusion, and the loss of pelvic articulation of the sacrum occur temporarily around the time of the change from fully terrestrial to fully aquatic locomotion during cetacean evolution (Thewissen et al., 2001) and rendered the hind limb ineffective on land. This change in environment from land to water impacted sacral morphology of extant cetaceans. Unlike early Eocene cetaceans, all modern cetaceans retain sacral intervertebral discs (Eschricht,

Reinhardt & Lilljeborg, 1866; Slijper, 1936; Buchholtz, 2001; Buchholtz,

Wolkovich & Cleary 2005; Buchholtz, 2007).

Cervical Intervertebral Fusion

Cervical intervertebral fusion occurs in some modern cetaceans and

Sirenians (Eschricht et al., 1866; Wheeler, 1930; Slijper, 1936; Van Valen, 1967;

Haldiman & Tarpley, 1993; Buchholtz, 2001; Buchholtz et al., 2005; Buchholtz,

2007; Buchholtz et al., 2007), but does not occur in most terrestrial mammals.

The number of fused cervical vertebrae in Mysticetes (baleen whales) and odontocetes (toothed whales) vary. Assessment of cervical intervertebral fusion stated in the past have been made on small samples; we therefore wanted to provide investigation of timing, degree, and pattern of cervical intervertebral fusion in an ontogenetic sample of bowhead whales (Balaena mysticetus). In adult bowheads, all seven cervical vertebrae fuse representing a novel morphology compared to terrestrial taxa. We also aim to understand the pattern of cervical intervertebral fusion in order to compare cervical fusion in cetaceans to sacral fusion in terrestrial mammals and examine whether cervical intervertebral fusion is correlated with age in bowhead whales.

Vertebral Epiphsyeal Fusion

Vertebral epiphyseal fusion has been studied in humans (Albert & Maples,

1995) and several species of cetaceans including both odontocetes (Ziphius sp.,

Mesoplodon ginkodens, Hyperoodon ampullatus, Moore, 1968); (Stenella coeruleoalba, Ito & Miyazaki, 1990); (Tursiops truncatus, Mead & Potter, 1990);

(Neophocaena phocaenoides, Yoshida et al., 1994); (Phocoena phocoena,

Cephalorhynchus commersonii, Lagenorhynchus albirostris, Galatius, 2010) and mysticetes (Balaenoptera physalus, Wheeler, 1930); (Balaenoptera acutorostrata, Kato, 1988); (Caperea marginata, Kemper & Leppard, 1999);

(Balaenoptera borealis, Best & Lockyer, 2002). These studies documented that epiphyseal fusion in modern cetaceans is delayed compared to most other mammals (Wheeler, 1930; Ohsumi et al., 1958; Kato, 1988; Ito & Miyazaki, 1990;

Yoshida et al., 1994; Kemper & Leppard, 1999; Best & Lockyer, 2002; Galatius,

2010), but they did not show evolutionary or developmental data. Delayed epiphyseal fusion has also been documented in other mammals as well, such as naked mole rats (Dengler-Crish & Catania, 2009), macaques (Nakai, 2001), and meerkats (Russell et al., 2004).

Mechanisms underlying the delay in epiphyseal fusion are unknown in modern cetaceans. The vertebral epiphyses fuse in a specific pattern, within a vertebra (the cranial and caudal epiphyses) and across the column from cervical to caudal vertebrae. Specifically, the cranial epiphysis fuses to the centrum before the caudal epiphysis in dolphins, (e.g. Stenella coeruleoalba Ito &

Miyazaki, 1990) and the epiphyses of the anterior thoracic vertebrae are the last to fuse (Wheeler, 1930; Ohsumi et al., 1958; Best & Lockyer, 2002). Previous studies have established epiphyseal fusion patterns in some extant cetacean taxa, but currently it is unclear if this pattern is present in archaic cetaceans. We aim to understand if epiphyseal fusion patterns are retained over evolutionary time by investigating and comparing early fossil and modern cetaceans.

To do this, I focus on two vertebral regions of normal fusion, the sacrum of terrestrial mammals and the neck of bowhead whales.

Materials and Methods

This study used an ontogenetic series of mice (Mus musculus, Linnaeus,

1758), n=20, skeletal samples of pig (Sus scrofa, Linnaeus, 1758), n= 9, anatomical specimens of bowhead (Balaena mysticetus, Linnaeus, 1758), n=8 and beluga (Delphinapterus leucas, Pallas, 1776), n=9 whales, pantropical spotted dolphin (Stenella attenuata, Gray, 1846), n= 2 fetuses, and fossil cetacean specimens, including Ambulocetus natans (Thewissen, Hussain & Arif,

1994), n= 1 and Kutchicetus minimus (Bajpai & Thewissen, 2000), n= 1. These species were compared to fossil taxa previously published. Techniques included

CT imaging, whole mount clearing and staining, anatomical dissections, and osteological study.

Ontogenetic Series of Mus musculus

We collected an ontogenetic postnatal mouse series at ten day intervals from birth (Day 1) to postnatal Day 60, and specimens older than one year.

Specimens were cleared and stained to show only bone (Alizarin Red) and cartilage (Alcian Blue) structures; all other tissues were chemically cleared, using a protocol adapted from Wassersug (1976). This series was collected following

IACUC standards at NEOMED, under protocol 09-020.

Artiodactyl Osteological Collection

Pigs were chosen as a comparison species because they have a generalized vertebral column and are members of Artiodactyla, the mammalian order that includes modern cetaceans (Thewissen & Madar, 1999; Geisler et al.,

2007; Thewissen et al., 2007; Spaulding, O’Leary & Gatesy, 2009). Osteological specimens were studied at Field Museum of Natural History in Chicago, IL and the Carnegie Museum of Natural History in Pittsburgh, PA. Ages of the pigs were estimated from tooth eruption and wear pattern (Getty, 1975; Hillson, 1986).

Modern Cetacean Osteological Samples

Osteology of cetacean patterns was documented in bowhead and beluga whales. Vertebral samples of bowhead (n= 8) and beluga (n= 9) whales were collected from specimens killed in subsistence hunts in Barrow and Point Lay,

Alaska. Vertebral counts and intervertebral and epiphyseal fusion patterns were recorded. Samples of vertebral centra, intervertebral discs, growth plates and epiphyses were cut using manual and power saws in the sagittal plane. Once harvested, bowhead and beluga cervical vertebrae samples were skeletonized.

All bowhead and beluga samples were collected under the NOAA-NMFS 814-

1899-01 permit.

Eocene Cetaceans

For analysis of fossil cetacean vertebrae, we chose two archaic cetaceans for which mostly complete vertebral columns are known. Ambulocetus natans

(Ambulocetidae, HGSP 18507; Thewissen, Madar & Hussain, 1996; Madar,

Thewissen & Hussain, 2002) is an amphibious cetacean (Bajpai & Thewissen,

2000; Madar et al. 2002) and Kutchicetus minimus (Remingtonocetidae, IITR-SB

2647; Thewissen & Bajpai, 2009; Bebej, 2011) is a semi-aquatic cetacean; both lived in near shore marine environments. These two cetaceans represent the most basal cetaceans for which much of the vertebral columns are known for a single individual.

CT scans

Computed tomography (CT) scans of modern and fossil cetacean specimens were completed at Loyola University Medical Center in Maywood, IL using a Siemens Somatom Sensation computed tomography scanner. This scanner produced sequential slices at .6 mm thickness. A Norland Stratec XCT

Bone Densitometer micro computed tomography scanner was used to scan small specimens at NEOMED, which scanned 11 images at 23 mm intervals and 0.25 mm thicknesses per vertebral centrum. CT images were studied using Image J

(Rasband, 2011).

Fusion Maps to Score Epiphsyeal Fusion

Epiphyseal fusion is illustrated in fusion maps showing fusion within a single vertebra and across the vertebral column in a diagram where each vertebra is represented by a rectangular box: a fusion map (Figs. 16-20). We used the degree of epiphyseal fusion to score vertebrae and distinguish three

66 states: 1) No fusion, a space is visible grossly or on CT scans between the centrum and epiphysis. In fossils and bony specimens, this space can be filled with mineral or air. In some specimens the epiphysis may also be loose and not present. In cleared and stained specimens, the cartilaginous growth plate is present between the epiphysis and the centrum (light grey, Figs. 16-20). 2)

Partial fusion, the epiphysis and the centrum are partially fused, evenly across the growth plate. An external fusion scar is a visible line on the surface of the bone or fossil between the epiphysis and the centrum (dark grey, Figs. 16-20).

3) Complete fusion, no remnant of fusion present (black, Figs. 16-20).

Fusion data for mice were collected from the cleared and stained ontogenetic mouse series. This technique provided clear visualization of the epiphyseal fusion pattern as the stain differentiates between bone and cartilage.

The bowhead whale fusion data was collected from sagittal cuts through the vertebral column. Historically, horizontal cuts were made on the ventral side of the vertebra to determine epiphyseal fusion pattern (Eschricht et al., 1866;

Wheeler, 1930; Ohsumi et al., 1958; Kato, 1988; Best and Lockyer, 2002).

Institutional Abbreviations

HGSP, Howard University- Geological Survey of Pakistan, Quetta,

Pakistan- fossils on loan to NEOMED, Rootstown, OH, USA; IITR-SB, Indian

Institute of Technology, Roorkee, India- Sunil Bajpai collection; NOAA-NMFS,

National Oceanic and Atmospheric Administration- National Marine Fisheries

Service.

Results

Sacral Intervertebral Fusion

Mus musculus

Intervertebral sacral joints fuse in a cranial to caudal direction in mice.

During ontogeny, the first sacral joint (S1S2) fuses first, followed by the second

(S2S3) and finally the third (S3S4) sacral joint. Intervertebral fusion causes sacral discs to decrease in width and eventually be replaced by bone (Fig. 14A-

C). At 20 days postnatal all intervertebral discs are intact (Fig. 14A). The nucleus pulposus (np) is present in each sacral joint. By 40 days postnatal the

S1S2 and S2S3 intervertebral discs stain with Alizarin red indicating the presence of bone (Fig. 14B and C). The S3S4 joint is maintained at this age and the nucleus pulposus is still intact (Fig. 14B). Residual growth plates (gp) are present as thin strips of blue stained cartilage (Fig. 14B and C). The S3S4 joint remains unfused past 60 days postnatal (Fig. 14C).

Cleared and stained mouse (Mus musculus) sacra, ventral view. A, 20 days; B, 40 days; C, 60 days postnatal. Ontogenetic skeletonized pig (Sus scrofa) sacra. D, 6-8 months (49844); E, 18-20 months (42439); F, >18-20 months (C3) postnatal. S1, S2, S3, S4, sacral vertebrae 1-4; gp, growth plate; np, nucleus pulposus; tp, transverse process; uf, unfused.

Figure 14. Land mammal sacra

Sus scrofa

In pigs, the S1S2 joint is the first to fuse and fusion progresses caudally to the last sacral joint, like in mice. At 6-8 months of age, sacral intervertebral fusion has started in the S1S2 and S2S3 joints, but not in the S3S4 joint; it is completely unfused and the S4 vertebrae is not present (Fig. 14D). By 18-20 months, the transverse processes (tp) of the sacral vertebrae are completely fused and intervertebral fusion is complete (Fig. 14E). This indicates that intervertebral fusion occurs before epiphyseal fusion in pigs. In pigs older than

20 months all sacral joints are fused (Fig. 14F).

Ambulocetus natans

CT scans of the Ambulocetus natans sacrum (HGSP 18507.834;

Thewissen et al., 1994; Thewissen et al., 1996) indicated fusion of four sacral vertebrae with fusion complete in the S1S2 and S2S3 joints (Fig. 15A). The

S3S4 joint is fused at the periphery of the joint and no external evidence of fusion is present. However, an oval- shaped radiolucent space (sp) is visible on the CT scan; this may be where the nucleus pulposus once resided (Fig. 15A and B).

There is a thin radio-opaque line visible on the CT scans between S3 and S4 extending from the edges of this oval space (Fig. 15A). This white line is a narrow extension of the air- filled space between S3 and S4 that was infiltrated by mineral and is unfused.

A, horizontal CT scan of Ambulocetus natans sacrum (HGSP 18507); B, sagittal CT scan of the Ambulocetus natans sacrum; C, ventral view of the Kutchicetus minimus sacrum (IITR-SB 2647), scale is 1 cm in length; D, coronal CT scan of the Kutchicetus minimus sacrum. gp, growth plate; sp, air-filled space; uf, unfused.

Figure 15. Archaic cetacean sacra

Kutchicetus minimus

The sacrum (IITR-SB 2647.13) of Kutchicetus minimus was collected in the field as four separate vertebrae. Thewissen and Bajpai (2009) deduced that

Kutchicetus was immature based on the incomplete fusion of its sacrum. The sacral vertebrae are only weakly synostosed and the space between each sacral vertebra is visible externally (Fig. 15C) and on the CT scan (Fig. 15D). Therefore the timing of intervertebral fusion pattern for Kutchicetus remains unknown.

Cervical Intervertebral Fusion

In modern bowhead whales, the seven cervical vertebrae fuse and eventually form a solid bony neck (Fig. 16). These bony bowhead specimens exhibit a slow rate of intervertebral fusion, which is already in progress by one year postnatal (Fig. 16A). Intervertebral fusion starts in the center of the vertebrae and progresses peripherally (Fig. 16A). This pattern of cervical fusion is opposite to the fusion pattern exhibited in the sacra of terrestrial mammals and

Eocene cetaceans. Fusion progresses across both the intervertebral discs and the epiphyses simultaneously (Fig. 16). By 40 years of age, all seven cervical vertebrae are completely fused and little differentiation of each vertebra is visible

(Fig. 16F). Cervical intervertebral fusion is not present at any age in beluga whales.

A, 1 yr old (09B11); B, 2 yrs old (10B16); C, 5 yrs old (11B8); D, 5-10 yrs old (09B9); E, ~20 yrs old (10B15); F, 20 yrs old (11B9); G, ~40 yrs old (11B3). Intervertebral fusion starts in the center of the Balaena mysticetus cervical vertebrae and progresses peripherally until the vertebrae are completely fused. Notice the fusion crosses the intervertebral disc as well as the epiphyseal plate. Scale bar: 1 cm for A-F. Scale bar: 5 cm for G.

Figure 16. Bowhead whale cervical specimens and cervical fusion maps

Epiphyseal Fusion

Mus musculus

The cleared and stained ontogenetic mouse series shows the cranial epiphysis always fuses to the centrum before the caudal epiphysis (Fig. 17).

Epiphyseal fusion starts around 10 days postnatal (Fig. 17B) and begins in the cervical vertebrae and progresses caudally towards the lumbar region (Fig. 17B-

F). Epiphyseal fusion starts in the sacrum simultaneously and progresses caudally towards to the tail (Fig. 17B-F). At 20 days postnatal, epiphyses of all seven cervical vertebrae are completely fused as well as the four sacral vertebrae (Fig. 17C). The cranial epiphyses of the thoracic vertebrae are completely fused, but the caudal epiphyses are not fused yet. The lumbar and seven anterior caudal epiphyses are partially fused. At 30 days postnatal the epiphyses of the anterior thoracic vertebrae are completely fused and the caudal vertebral epiphyses are partially fused (Fig. 17D). By 40 days postnatal the cervical, sacral, and anterior caudal vertebrae are completely fused as are the cranial epiphyses of most of the posterior caudal vertebrae (Fig. 17E). The lumbar caudal epiphyses are the last to fuse (Fig. 17F). Complete epiphyseal fusion is present by 60 days postnatal (Fig. 17G and H).

Sus scrofa

Like in mice, the cranial epiphysis always fuses to the centrum before the caudal epiphysis in Sus scrofa. Epiphyseal fusion starts in the cervical vertebrae

A, 1 day (M81); B, 10 day (M54); C, 20 day (M82); D, 30 day (M78); E, 40 day (M143); F, 50 day (M83); 60 day (M84); 230 day (M85) postnatal Mus musculus specimens. Epiphyseal fusion starts in the cervical vertebrae and in the sacral vertebrae and progresses caudally. The lumbar epiphyses are the last to fuse (F). C2, second cervical vertebra; T1, first thoracic vertebra; L1, first lumbar vertebra; S1, first sacral vertebra; Ca1, first caudal vertebra. Light gray, no epiphyseal fusion; dark gray, partial epiphyseal fusion; black, complete fusion, white, missing vertebra.

Figure 17. Mouse postnatal ontogenetic fusion maps

75 and progresses caudally (Fig. 18). Simultaneously, epiphyseal fusion begins in the sacrum and progresses cranially and caudally (Fig. 18E). Epiphyseal fusion continues in two directions within the column leaving the anterior thoracic vertebral epiphyses to fuse last (Fig. 18 H and I).

Ambulocetus natans

In Ambulocetus natans, the cranial epiphyses of the last cervical vertebra and first thoracic vertebra, all four sacral vertebrae, and the anterior caudal epiphyses are completely fused (Fig. 19A). Most of the thoracic and lumbar vertebrae exhibit partially fused epiphyses (Fig. 19A). Epiphyseal fusion in the posterior caudal vertebrae is unknown as is the exact number of tail vertebrae.

The cranial epiphysis fuses before the caudal epiphysis like in mice and pigs

(Fig. 19A). In Ambulocetus natans the overall epiphyseal fusion pattern is consistent with starting at the cranial and caudal ends of the vertebral column and epiphyseal fusion progresses towards mid-column. The posterior thoracic vertebrae and the anterior lumbar vertebrae are the last to fuse.

Kutchicetus minimus

Epiphyseal fusion is complete in most cervical, sacral, and caudal vertebrae in Kutchicetus minimus (Fig. 19B). The epiphyses of S1, S2, and S3 are completely fused externally, however, internal morphology from CT scans show only the cranial and caudal epiphyses of S1 and the cranial epiphysis of S2 are completely fused; the other sacral epiphyses are partially fused (Figs. 19B

A, 49844; B, C2; C, 42440; D,42439; E, C1; F, C3; G, 97884; H, 92908; I, 92907. Relative age of Sus scrofa individuals was determined using dental eruption, tooth wear, and epiphyseal fusion. Eruption of the second (M2) and third (M3) molars are provided. Epiphyseal fusion starts in the cervical vertebrae and the sacral region and progresses cranially and caudally. The thoracic region is the last to fuse. Light gray, no epiphyseal fusion; dark gray, partial epiphyseal fusion; black, complete fusion; white, missing vertebra.

Figure 18. Pig postnatal ontogenetic fusion maps

A, Ambulocetus natans (HGSP 18507); B, Kutchicetus minimus (IITR-SB 2647). Epiphyseal fusion starts in the cervical and caudal vertebrae and progresses towards the middle of the column. Epiphyses with questionable fusion are marked with questions marks (?). The exact number of tail vertebrae in these species is unknown. Light gray, no epiphyseal fusion; dark gray, partial epiphyseal fusion; black, complete fusion; white, missing vertebra.

Figure 19. Archaic cetacean fusion maps

78 and 2D). They are separated from the centrum by a radio-opaque line that represents the growth plate space (Fig. 15D). The cranial epiphyses of all

Kutchicetus vertebrae fuse prior to the caudal epiphyses like in mice, pigs, and

Ambulocetus. The overall epiphyseal fusion pattern in the Kutchicetus minimus vertebral column is consistent with starting in the cervical and caudal regions of the column and progressing towards the middle of the column as exhibited in

Ambulocetus.

Modern Whales

In bowhead whales, epiphyseal fusion starts in the cervical vertebrae and continues in a caudal direction while simultaneously fusion in the caudal vertebrae progresses cranially (Fig. 20A-D). Fusion progressing from the caudal vertebrae seems to advance at a slower rate than fusion originating in the cervical region (Fig. 20A-D). The cervical epiphyseal fusion is not complete in a one year old bowhead whale (Fig. 20A). The cranial and caudal epiphyses of cervical vertebrae 3-7 are partially fused (Figs. 16 and 20) as are the epiphyses of the anterior caudal vertebrae. The epiphyses of the cervical and anterior thoracic vertebrae are completely fused in a five year old bowhead (Fig. 20B and

C). The thoracic and lumbar vertebral epiphyses remain unfused through 20 years of age (Fig. 20D). The skeletonized cervical samples show epiphyseal fusion progresses at a faster rate than cervical intervertebral fusion suggesting intervertebral fusion is complete after epiphyseal fusion (Fig. 16). Beluga whales

Balaena mysticetus specimens: A, 1 yr (09B11); B, 5 yrs (11B8); C, 5-10 yrs (09B5); D, 20 yrs (11B9). The ages of these individuals are determined from baleen length and retinal chemistry. Delphinapterus leucas specimens: E-M. Individual belugas are ranked according to epiphyseal fusion, ages unknown. Epiphyseal fusion starts in the cervical and caudal vertebrae and progresses towards the middle of the column. Light gray, no epiphyseal fusion; dark gray, partial epiphyseal fusion; black, complete fusion; white, missing vertebra.

Figure 20. Bowhead and beluga whale fusion maps

80 show the same overall pattern of epiphyseal fusion (Fig. 20E-M) as bowhead whales. Epiphyses of the cervical and caudal vertebrae fuse first and fusion progresses towards mid-column.

Discussion

The morphological changes in the cetacean vertebral column, pelvis, and hind limb are associated with the habitat change from land to water and in particular locomotor style. There are four main morphological changes occurring in the evolution of cetaceans in regard to the pelvic girdle and hind limb (Fig. 21):

1) reduction of fusion between sacral vertebrae, 2) loss of articulation between the sacrum and the pelvis 3) reduction of hind limb, and 4) loss of intrapubic articulation in the pelvis. All four of these morphological changes directly impact locomotor style and will be discussed.

Evolution of the Sacral Morphology in Cetaceans

In evolution, sacral intervertebral fusion is lost first between the S3 and S4 vertebrae and the lack of fusion progresses cranially until S1 is the only sacral vertebrae to articulate with the pelvis (Fig. 22). The changes in intervertebral fusion during the evolution of cetaceans begin in artiodactyls, the sister group of modern cetaceans (Thewissen et al., 2001).

Representing the primitive artiodactyl condition in sacral morphology are two terrestrial basal artiodactyls, Diacodexis from North America (Rose, 1985)

A- articulation between sacral vertebrae, B- articulation between the sacrum and pelvis, C- articulation between the pelvis and hind limb, D- pubic symphysis articulation.

Figure 21. Four articulation points altered by evolution mapped on an alpaca pelvis

Notice the evolutionary loss of intervertebral fusion, or the decrease in the number of fused sacral verterabrae, from primitive artiodactyls (Diacodexis and Gujaratia) to modern cetaceans. Also, the loss of vertebral articulation with the pelvis can be traced evolutionarily. These morphological changes occur in parallel with locomotor style (terrestrial, waders, amphibious, semi-aquatic, aquatic) and environment from land to water.

Figure 22. Cetacean phylogeny of sacral fusion (based on Geisler et al., 2007; Geisler and Theodor, 2009; and Spaulding et al., 2009)

83 and Gujaratia from Pakistan (Bajpai et al., 2005; Theodor, Erfurt & Metais, 2007).

Both basal artiodactyls have a three vertebrae fused sacra (Thewissen &

Hussain, 1990).

Inferring from the sacrum of the Raoellid Indohyus, the semi-aquatic artiodactyl and close relative to cetaceans with a disputed exact phylogenetic position (Geisler et al., 2007; Geisler & Theodor, 2009; Spaulding et al., 2009), we think it is possible that Diacodexis and Gujaratia are juveniles and this is the reason there is a lack of intervertebral fusion between S3 and S4 in both specimens. In the case of Indohyus, a total of four sacral vertebrae were recovered, three of which are fused (Thewissen et al., 2007; Cooper et al., 2012).

The fourth sacral vertebrae was found unfused from the sacrum (Cooper et al.,

2012), but this may be due to ontogeny and not morphology.

Pakicetids, the earliest whales (Gingerich & Russell, 1981; Williams, 1989;

Madar, 2007; Cooper el al., 2009) had a sacrum consisting of four fused vertebrae. Ambulocetus natans (HGSP 18507- Madar et al., 2002) and

Kutchicetus minimus (IITR-SB 2647- Thewissen & Bajpai, 2009) were semi- aquatic living in near shore marine environments (Bajpai & Thewissen, 2000;

Madar et al., 2002). Both taxa had four fused sacral vertebrae, as did Dalanistes ahmedi (Gingerich, Arif & Clyde, 1995) and Remingtonocetus domandaensis

(Gingerich et al., 2001; Bebej, 2012).

The last sacral vertebra (S4) is not fused in Maiacetus inuus; this may be due to this individual being a juvenile and S4 is not yet fused (Gingerich et al.,

2009). The loss of sacral fusion continues with a two vertebrae fused sacrum in

Qaisracetus arifi (Gingerich et al., 2001), Makracetus bidens, (Gingerich et al.,

2005), and Takracetus simus (Gingerich et al., 2001). Takracetus simus morphology is based on Gingerich et al. (2001) and not primary analysis. In

Protocetus atavus, there is no articulation between the S1 and S2 vertebrae

(Buchholtz, 1998); this lack of intervertebtral fusion is present in Rodhocetus kasrani (Gingerich et al., 1994); Protocetus atavus, Fraas, 1904; Kellogg, 1936;

Gingerich et al., 1993; Buchholtz, 1998); Natchitochia jonesi (Uhen, 1999);

Gaviacetus razai (Gingerich et al. 1995).

The loss of intervertebral fusion in the sacrum of cetaceans may have allowed for more flexibility in dorsoventral undulation during swimming.

Increased spinal flexibility during swimming may have provided a larger range of motion and therefore an increase in swimming speed. By prioritizing fully aquatic body morphology, swimming efficiency may have increased.

Evolution of Pelvic Articulation in Cetaceans

In terrestrial mammals and early Eocene cetaceans the articulation of the pelvis to the vertebral column anchors the hind limb to the axial skeleton providing stability during terrestrial locomotion (Gingerich et al., 1990; Mulder,

2001; Buchholtz, 2007; Adam, 2009).

Sacral articulation with the pelvis is present (Fig. 22) in artiodactyls and basal cetaceans, but it is lost higher in the cladogram, possibly independently

85 multiple times such as in Makracetus bidens (Gingerich et al., 2005) and

Takracetus simus (Gingerich et al. 2001). The lack of pelvic-vertebral articulation is characteristic of late protocetids and basilosaurids (Gingerich et al., 1990;

Buchholtz, 2007; Adam, 2009) specifically, Georgiacetus vogtlensis (Hulbert et al., 1998), Dorudon atrox (Uhen, 2004) and Basilosaurus isis (UM 97525,

Gingerich et al., 1997; Uhen, 1999).

The loss of sacral articulation with the pelvis increased flexibility in the lower spine and pelvic girdle and supported the flexibility already increased by the lack of fusion between sacral vertebrae. The lack of pelvic articulation with the spine suggests that later archaic cetaceans were unable to support their body weight on land and their locomotor style was evolving from fully terrestrial to fully aquatic.

Lack of articulation between the pelvis and hind limb loosened the pelvic girdle. This enlargement may have been associated with having larger, precocial offspring. For example, a Maiacetus specimen (GSP-UM 3475a) was recovered with a fetus (GSP-UM 3475b) still in the womb (Gingerich et al., 2009).

Gestational orientation of the fetus was with the rostrum pointing caudally; this suggests this fetus was near fully term (Gingerich et al., 2009). This is the same birth orientation displayed in modern cetaceans that also have disarticulation of the pelvis and sacrum. This interpretation of this fossil is different according to Thewissen and McClellan (2009) who state Maiacetus

(GSP-UM 3475a) probably ate GSP-UM 3475b) and that is why they are in this associated orientation.

Evolution of Reduced Hind Limb in Cetaceans

The reduction of the hind limb in cetaceans can be discussed in a developmental and evolutionary context. Developmentally, dolphins form hind limb buds around the fifth gestational week and later the buds are resorbed before birth (Thewissen et al., 2006; Thewissen et al., 2009). Similarly to retaining molecular signaling for the formation of a hind limb, cetaceans retain molecular signaling for the sacrum as well though intervertebral fusion never occurs in the sacral region (Fig. 23).

Early archaic cetaceans, Ambulocetus and Remingtonocetus, had hind limbs that articulated with the pelvis at the acetabulum suggesting the hind limb played a significant role in their amphibious locomotion (Hulbert et al., 1998).

From the morphology and articulation it is clear their hind limbs were weight bearing during terrestrial locomotion, but also played an active role in their swimming style. In Ambulocetus the hind limb was used to paddle through water during aquatic locomotion (Madar et al., 2002). The hind limb is greatly reduced in basilosaurids though they retain a femur, tibia, and fibula; the basilosaurid hind limb is unable to bear weight suggesting basilosaurids were fully aquatic

(Gingerich et al., 1990; Uhen, 1993; Thewissen & Fish, 1997).

Notice the four dark triangular ossifications in the sacral region in A- LACM 94310. These ossifications are larger in B- LACM 94382 and they are larger than those ossifications in adjacent vertebrae. These ossifications may suggest modern cetaceans retain molecular signaling for a sacrum though sacral intervertebral fusion does not occur.

Figure 23. Cleared and stained Stenella attenuata

The reduction of hind limbs in cetaceans may contribute to lessening drag during swimming (Bejder & Hall, 2002). Some modern mysticetes, for example, the bowhead whale, retain a pelvis, femur, and tibia (Eschricht et al., 1866;

Haldiman & Tarpley, 1993; Thewissen et al., 2009) and these vestiges reside in the anterior abdominal wall (Thewissen et al., 2009) and are therefore not factors in drag. With the reduction of the hind limbs swimming style became reliant upon the tail for propulsion (Fish, 1996; Fish et al., 2003).

Among mammals the reduction of hind limbs is unique to cetaceans, but reduction of hind limbs has evolved multiple times. Snakes and limbless lizards exhibit this morphology as well (Gans, 1975; Cohn & Tickle, 1999; Bejder & Hall,

200). These animals have a reduced hind limb, elongation of the vertebral column, and loss of regionalization in the vertebral column (Cohn & Tickle, 1999;

Bejder & Hall, 2002) all of which are exhibited in modern cetaceans. Snakes, evolved from tetrapods closely related to mosasaurs (Cohn & Tickle, 1999), retaining a reduced pelvis and short femur located near the cloaca. A change in

Hox gene expression or regulation, responsible for axial patterning, may be responsible for the reduced pelvic morphology exhibited in snakes and limbless lizards (Cohn & Tickle, 1999; Bejder & Hall, 2002). It is possible that Hox expression in cetaceans is responsible for the absence of hind limbs in adult cetaceans, but this was not included in the scope of this project.

Evolution of Intrapubic Articulation and Reduction of the Pelvis in Cetaceans

The overall reduction of the pelvis is thought to be associated with the loss of abdominal muscle insertion on the pelvis (Simões-Lopez & Gutstein, 2004;

Adam, 2006; Thewissen et al., 2006; Bebej, 2012). The pelvis in modern whales, although not functional in locomotion, acts as a site for muscle and genitalia attachment (Simões-Lopez & Gutstein, 2004; Adam, 2009; Thewissen et al.,

2009). Though the pelvis does not articulate with the sacrum these structures do articulate with each other (Eschricht et al., 1866; Haldiman & Tarpley, 1993;

Thewissen et al., 2009).

Pelvic reduction in cetaceans includes loss of the public symphysis.

Disarticulation of the pubic symphais can be associated with the expansion of the pelvic cavity by allowing the reduced pelvis to be located laterally. The loss of the pubic symphysis may be associated with the reduction of the hind limb and the reduction of associated musculature (Bebej, 2012).

Cervical Intervertebral Fusion

The distribution of cervical intervertebral fusion is well understood in modern cetaceans (Fig. 24), but does not occur in Eocene cetaceans. Plotting cervical fusion and neck length on a cetacean cladogram (Messenger &

McGuire, 1998), it appears that shorter necks tend to be fused like that of the bowhead whale while necks with longer lengths are unfused (Fig. 24). An important factor in understanding cervical intervertebral fusion is ontogeny. Our

This plot illustrates the relationship between neck length and cervical intervertebral fusion. Shorter necks tend to have two or more cervical vertebrae fused. White boxes- 0 fused cervical vertebrae, grey boxes- 2-4 fused cervical vertebrae, black boxes- 6-7 fused cervical vertebrae. Phylogeny based on Messenger and McGuire (1998) strict consensus phylogeny, cervical fusion counts (Wheeler, 1930; Slijper, 1936; Haldiman and Tarpley, 1993; Buchholtz, 2001; Buchholtz et al., 2005, Buchholtz 2007; Buchholtz, 2010), Remingtonocetus (Bebej et al., 2012), Dorudon (Uhen, 2004).

Figure 24. Phylogeny of cervical fusion related to neck length

91 data on bowhead whale shows cervical fusion to arise late in life, which indicates that large samples of whales are needed to determine the amount of cervical fusion. If I had only studied one or two individuals, I may not have known that it takes possibly forty years to complete cervical intervertebral fusion. Therefore one should be careful in interpreting cetacean data based solely on a single individual.

In the bowhead whale, cervical vertebrae fusion starts centrally and progresses peripherally (Fig. 16) within the intervertebral disc. The first region to undergo ossification is the nucleus pulposus and the last region is the annulus fibrosus (Fig. 16). This fusion pattern is different from the intervertebral fusion pattern exhibited in terrestrial mammal sacral formation. Intervertebral fusion in the terrestrial sacrum starts in the annulus fibrosus and endplates of the intervertebral disc progressing towards the center of the joint where the nucleus pulposus is located (Fig. 14). The functional significance for cervical intervertebral fusion is unknown. It could be that cervical intervertebral fusion occurs in response to maintaining the position of the large head in a streamlined orientation for more efficient swimming (Buchholtz, 2001).

Another functional reason for cervical intervertebral fusion may be associated with the inner ear. The semicircular canals are located in the inner ear and are part of the organ of balance providing information on spatial orientation and movement (Spoor, 2003). Semicircular canal diameter tends to scale allometrically with body size in terrestrial taxa (Spoor, 2003); large bodied

92 animals typically have large semicircular canals. I overlaid semicircular canal size on the phylogeny for neck length and cervical intervertebral fusion (Fig. 25) to determine if there are any associations between semicircular canal size and neck morphology in cetaceans.

Surprisingly, modern cetaceans have tiny semicircular canals relative to body size (Ketten, 1992; Spoor et al., 2002), though bowhead whales have the largest semicircular canals among cetaceans (Spoor et al., 2002). A stiff neck, like that of bowhead whales, impacts the balance control of the semicircular canals by limiting three-dimensional sensory input (Ketten, 1992), but really a fused neck would render semicircular canals useless because independent sinusoidal head rotation is reduced to nothing in bowhead whales. Perhaps cetaceans with small semicircular canals and fused cervical vertebrae are the intermediate in the evolution of semicircular canals and eventually they will be vestigial.

Epiphyseal Fusion

Epiphyseal fusion occurs at the cranial and caudal end of each vertebra similarly to the long bones of the appendicular skeleton. In both modern terrestrial mammals and Eocene cetacean epiphyseal fusion starts in two locations in the vertebral column (Fig. 4-6): in the cervical vertebrae and in the sacrum and fusion progresses caudally from both vertebral regions. In pigs the sacral fusion progresses cranially as well as caudally (Figs. 4-6). In Eocene

This plot illustrates the relationship between semicircular canal size, neck length, and cervical intervertebral fusion in cetaceans. From this plot we are unable to determine a relationship between cervical morphology and semicircular canal size in cetaceans. Semicircular canal size data (Spoor and Thewissen, 2008).

Figure 25. Semicircular canal size overlaid on the cervical fusion and neck length data

94 cetaceans, epiphyseal fusion starts in the cervical and caudal vertebrae and fusion progresses towards mid vertebral column (Fig. 3). This is consistent with the epiphyseal fusion pattern exhibited in bowhead and beluga whales (Fig. 7) as well as other cetacean species (Balaenoptera physalus, Wheeler, 1930; Ohsumi et al., 1958; Balaenoptera acutorostrata, Kato, 1988; Stenella coeruleoalba; Ito &

Miyazaki, 1990; Galatius, 2010; Neophocaena phocaenoides, Yoshida et al.,

1994; Balaenoptera borealis, Best & Lockyer, 2002; Phocoena phocoena,

Galatius & Kinze, 2003).

Modern bowheads retain open vertebral growth plates past 20 years of age; this was observed in the lumbar vertebrae long after sexual and skeletal maturity based on the first corpus luteum in the ovary and body length respectively (Chittleborough, 1955). Therefore, epiphyseal fusion patterns exhibited in modern whales are different from the epiphyseal fusion patterns exhibited in terrestrial mammals, as I have shown specifically in mice and pigs.

These differences in epiphyseal fusion may be related to different locomotor pattern exhibited by terrestrial and fully aquatic mammals.

Most likely there is a relationship between unfused epiphyses and the low core body temperature (33.6oC) of bowhead whales (George, 2009). The core temperature of bowhead whales is a few degrees lower than other mammals

(George, 2009). Previous studies have shown that bone growth and nutrient transport are affected by changes temperature (Serrat et al., 2008; Serrat et al.,

2009). When limbs were exposed to cool temperatures bones were shorter in

95 length and nutrient transport was slowed on its progression through the growth plate by adjacent blood vessels (Serrat et al., 2008; Serrat et al., 2009). Perhaps bowhead whales retain unfused growth plates to maintain nutrient diffusion to cartilage while living in water that is 0oC (George, 2009).

Conclusion

Sacral vertebrae fuse in a cranial to caudal pattern in terrestrial mammals like mice, pigs, and some Eocene whales such as Ambulocetus natans and

Kutchicetus minimus. Throughout whale evolution, the number of fused sacral vertebrae decreases from four in the Eocene to none in modern cetacean descendants. Along with the decrease in the number of fused sacral vertebrae, the loss of articulation with the pelvis is a unique morphology to cetaceans and sirenians. My results confirm sacral intervertebral fusion pattern is retained evolutionarily and modern cetaceans do exhibit some retained developmental signal in the sacral region that causes these vertebrae to ossify earlier than adjacent vertebrae.

Intervertebral fusion was not completely lost in modern cetaceans. Some modern cetacean species fuse the cervical vertebrae forming a solid bony neck.

Cervical fusion begins near the nucleus pulposus at the center of the intervertebral joint and progresses peripherally ontogenetically, as we have shown in bowhead whales. This fusion pattern could be used to age bowhead whales, but logistical issues prevent it from being an efficient aging technique.

We found epiphyseal fusion patterns differ between terrestrial and aquatic mammals. In terrestrial mammals, including Eocene cetaceans, epiphyseal fusion occurs in the cervical and sacral vertebral regions first. In modern cetaceans, epiphyseal fusion starts in the cervical and caudal vertebrae simultaneously and progress towards the middle of the vertebral column (Fig. 7).

These epiphyseal fusion patterns are most likely associated with locomotor and sensory anatomy. The epiphyseal fusion pattern exhibited by Eocene cetaceans is not retained in all modern mammals, but is present in modern cetaceans (Figs.

6 and 7).

CHAPTER VI

INTERVERTEBRAL FUSION IN CETACEAN (BALAENA MYSTICETUS) CERVICAL SPINE COMPARED TO INTERVERTEBRAL SACRAL FUSION IN TERRESTRIAL MAMMALS

Introduction

The intervertebral disc is comprised of three cartilaginous regions; the annulus fibrosis, endplate, and nucleus pulposus (Fig. 26). The annulus fibrosus is the outermost layer of the intervertebral disc composed of tough concentric lamellae of type I collagen-rich fibrocartilage (Antoniou et al., 1996; Roughley,

2004; Shankar et al., 2009; Risbud et al., 2010; Hristova et al., 2011; Smith et al.,

2011) The annulus fibrosus inserts into the endplates (Coventry et al., 1945;

Dahia et al., 2009), which are two regions of hyaline cartilage located at the cranial and caudal ends of each intervertebral disc, adjacent to the vertebral bodies (Coventry et al., 1945; Roberts et al., 1989, Moore, 2006; Shankar et al.,

2009; Smith et al., 2011). The endplates are characterized by hypertrophic chondrocytes (Dahia et al., 2009). Between the endplates, the nucleus pulposus is located in the center of the intervertebral disc (Fig. 26). The nucleus pulposus is composed of type II collagen and proteoglycans and is surrounded by the annulus fibrosus (Oegema, 1993; Antoniou et al., 1996; Dahia et al., 2009;

Hristova et al., 2011). 97 98

Horizontal section of a generalized intervertebral disc with all regions labeled.

Figure 26. Intervertebral disc diagram

In specific regions of the vertebral column, components of the intervertebral disc degenerate, disc cells apoptose (Ariga et al., 2001; Zhao et al.,

2006; Bertram et al., 2009), and adjacent vertebrae fuse together or synostosis

(Williams et al., 1995; Broome et al., 1998; Passalacqua, 2009). Vertebrae synostosis provides stability and strength to the vertebral column (Boden and

Schimandle, 1995). The location of intervertebral synostosis varies in the mammalian vertebral column (Williams et al., 1995; Buchholtz, 1998). The sacrum of terrestrial mammals is one vertebral region that fuses during normal ontogeny (Evans, 1993; Williams et al., 1995; Bab et al., 2007). The sacrum is comprised of multiple vertebrae and is the dorsal portion of the pelvis stabilizing the hind limb and distribution of forces through the hind limb (Hyman, 1922;

Williams et al., 1995).

A fused sacrum is present 50 million years ago in early Eocene cetaceans

(Thewissen et al., 2001; Gingerich, 2003; Bajpai et al., 2009; Thewissen et al.,

2009). During the land to water transition, in the Eocene, cetacean vertebral and pelvic morphology underwent major morphological changes (Thewissen et al.,

2001; Gingerich, 2003; Bajpai et al., 2009; Thewissen et al., 2009). These evolutionary changes in the cetacean vertebral column include reduction of the terrestrial sacrum to a single vertebra, eventual loss of pelvic articulation, and reduction of the hind limb (Thewissen et al., 2001; Gingerich, 2003; Bajpai et al.,

2009; Thewissen et al., 2009). The loss of sacral fusion, pelvic- vertebral joint articulation, and reduction of the hind limb correspond to environmental changes

100 and induced locomotor changes from fully terrestrial to fully aquatic swimming

(Thewissen et al., 2001). Due to these morphological changes all modern cetaceans as they retain the sacral intervertebral discs (Eschricht et al., 1866;

Slijper, 1936; Buchholtz, 2001; Buchholtz et al., 2005; Buchholtz, 2007).

Intervertebral fusion is not lost in Cetacea however; unusual patterns of intervertebral fusion occur in some cetaceans and manatees. In these aquatic mammals, sacral vertebrae never fuse, but cervical vertebrae do fuse (Eschricht et al., 1866; Wheeler, 1930; Slijper, 1936; Van Valen, 1967; Haldiman and

Tarpley, 1993; Buchholtz, 2001; Buchholtz et al., 2005; Buchholtz, 2007;

Buchholtz et al., 2007). Cervical intervertebral fusion does not occur in most terrestrial mammals and therefore cetaceans present a unique vertebral morphology among mammals. Mysticetes (baleen whales) and odontocetes

(toothed whales) exhibit a variable number of fused cervical vertebrae (Wheeler,

1930; Slijper, 1936; Stewart and Stewart, 1989; Haldiman and Tarpley, 1993;

Buchholtz, 2001; Buchholtz et al., 2005; Buchholtz, 2007; Buchholtz, 2010). In one species of mysticete, bowhead whale (Balaena mysticetus), all seven cervical vertebrae fuse representing a novel morphology compared to terrestrial taxa.

The cervical vertebrae of cetaceans are also unique because they are foreshortened in the cranial-caudal dimension (Romer, 1933; Buchholtz, 1998).

The neck of a bowhead whale is shorter than expected when compared to body length. Shortening the modern cetacean neck stiffens this vertebral region,

101 enhances the hydrodynamic shape of the body, and stabilizes the head and torso during fluke or tail propelled locomotion (Buchholtz, 1998; Fish, 2002; Fish et al.,

2003).

I aim to understand if the patterns of intervertebral fusion protein expression are the same in mammals regardless of fusion location within the vertebral column. To pursue this aim, I have two hypotheses;

1) The pattern of protein expression exhibited in cervical fusion in some

cetaceans, specifically bowhead whales is the same in sacral

intervertebral fusion of terrestrial mammals.

2) The use of cervical intervertebral fusion can be used as an aging

technique for bowhead whales.

To pursue these two hypotheses, I used immunohistochemistry to elicit protein expression in the cervical intervertebral discs of bowhead whales and analyzed skeletonized cervical bony samples to assess changes in cervical vertebrae morphology specifically the amount of intervertebral fusion to identify a correlation with age. I hope this trait can be added to aging techniques already in place and applied to bowhead whales.

Methods and Materials

Tissue Preparation

A postnatal ontogenetic series of bowhead whale (Balaena mysticetus) from the age of 1 year to 40 years (Table 1) was collected in Barrow, Alaska.

102

This series consists of both sexes and collection was in collaboration with the

Department of Wildlife Management, North Slope Borough under the NOAA-

NMFS 814-1899-01 permit with permission from the Alaska Eskimo Whaling

Commission and local subsistence hunters.

Table 1. Bowhead Whale (Balaena mysticetus) Samples

Specimen # Age Cervical Samples 09B5 5-10 yrs C1-5 09B9 5-10 yrs C1-7 09B11 1 yr C1-5 10B15 >20 yrs C1-5 10B16 2 yrs C1-7 11B3 40 yrs

Sagittal cervical sections were harvested using hand and mechanical tools. These samples include vertebral centrum, intervertebral disc, and growth plate. These vertebral samples were collected for immunohistochemistry to determine protein expression patterns. Each sample was fixed in 3% paraformaldehyde for approximately 2 weeks depending on the size of the sample. Vertebral samples were then decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 2-3 months or more depending on the size and age of the animal. After decalcification, the vertebral samples were washed in 1XPBS and specific joint samples were cut to a size appropriate for tissue processing. Samples were dehydrated in graded ethanol and xylene washes and embedded in Fisherbrand Paraplast X-tra paraffin. All vertebral

103 samples were embedded and cut in the horizontal plane at six microns using a

Leica RM 2165 microtome. Sections were mounted on Superfrost Plus microscope slides (Fischer Scientific).

Immunohistochemistry

Protein expression of GDF5, BMP2/4, MMP13, and TIE2 in the intervertebral disc was assessed using immunohistochemistry. After deparaffinization and rehydration in graded ethanol and xylene washes, antigen retrieval was completed using 85C sodium citrate for 10 minutes. Primary antibody concentration for GDF5 and MMP13 is 1:250; for TIE2 and BMP2/4

1:50 (Santa Cruz Biotechnology Inc). All primary antibodies were incubated at

4C overnight. Localization of the primary antibody was completed with the ABC anti-goat (sc-2023) or ABC anti-rabbit (sc-2018) staining kit (Santa Cruz

Biotechnology) following manufacturer’s protocol. Slides were counterstained using .01% thionin and coverslipped using DPX paramount. All experiments included a control slide which omitted the primary antibody.

Skeletonized Specimens

Osteology of cetacean cervical fusion patterns was documented in bowhead whales. Vertebral samples of bowhead whales (n= 8) were collected from specimens killed in subsistence hunts in Barrow, Alaska. Intervertebral fusion patterns were recorded. Samples include vertebral centra, intervertebral discs, growth plates and epiphyses. Samples were cut using manual and power

104 saws in the sagittal plane at midline. Once harvested, bowhead cervical vertebrae samples were skeletonized

CT Scans

Computed tomography (CT) scans of a one year old bowhead whale

(09B11) neck sample was completed at Loyola University Medical Center in

Maywood, IL using a Siemens Somatom Sensation computed tomography scanner. This scanner produced sequential slices at .6 mm thickness. CT images were studied using Image J (Rasband, 2011).

C & S Bowhead

One fetal Balaena mysticetus (n= 1) was cleared and stained to show only bone (Alizarin Red) and cartilage (Alcian Blue) structures; all other tissues were chemically cleared, using a protocol adapted from Wassersug (1976). Staging of the fetus is based on the Carnegie system adapted for Stenella attenuata by

Thewissen and Heyning (2007) and fetal age is based on Stěrba et al., (2000).

This bowhead fetus is from Carnegie stage 23.

Immunohistochemistry Results

MMP13 protein expression in the two year old bowhead (10B16, Fig. 27), is absent in intervertebral discs C1 through C6 because they are undergoing remodeling and are in the process of fusing (Fig. 27). The C6C7 intervertebral disc is not fused, but does not exhibit MMP13 protein expression (Fig. 27).

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A two year old (10B16), a 5-10 year old (09B9), and a 20 year old (10B15) bowhead whale. Key for the fusion map in Figure 16. Red X- no expression, Sagittal sections 40x immunohistochemistry pictures to show intracellular expression in bowhead whale cervical samples. Brown represents positive staining for MMP13.

Figure 27. MMP13 protein expression in bowhead cervical intervertebral discs

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At 5-10 years of age (09B9), MMP13 is absent in the C1 through C5 intervertebral discs (Fig. 27) and C5 through C7 endplate chondrocytes present

MMP13 protein expression (Fig. 27). At 20 years old (10B15) the C1C2 disc exhibits MMP13 protein expression in the cytoplasm of endplate chondrocytes

(Fig. 27). The C2 through C5 intervertebral joints are fused and MMP13 protein expression is absent (Fig. 27). Ontogenetically, MMP13 protein expression is maintained.

TIE2 protein expression in the two year old (10B16, Fig. 28) is absent from

C1 through C5 intervertebral discs; these joints are in the process of fusing. The

C6C7 intervertebral disc is intact and endplate chondrocytes express TIE2 in the cytoplasm (Fig. 28). TIE2 protein expression is absent from the C1 to C3 intervertebral disc of the 5-10 year old bowhead whale, but present in the C3C4 intervertebral disc (09B9, Fig. 28). TIE2 is absent in the C4 through C6 intervertebral discs and present in the C6C7 disc (Fig. 28). By 20 years of age

(10B15), TIE2 protein expression is absent in C1 through C5 and present in the

C6C7 disc (Fig. 28). Ontogenetically TIE2 protein expression is maintained

GDF5 protein expression is absent from C1 through C5 intervertebral discs at two years of age (10B16). These joints are undergoing intervertebral fusion. In the C6C7 intervertebral disc TIE2 protein expression is present in the cytoplasm of endplate chondrocytes (Fig. 29). In 09B9 (5-10 year olds), GDF5 protein expression absent from C1 through C3 and these joints are fusing. GDF5 is present in C4 through C7 and it is present regardless of the presence of fusion

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Figure 28. TIE2 protein expression in bowhead cervical intervertebral discs

108

Figure 29. GDF5 protein expression in bowhead cervical intervertebral discs

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(Fig. 29). By 20 years of age (10B15), there is an increase in GDF5 protein expression and is only absent in the C2C3 intervertebral disc (Fig. 29).

BMP2/4 protein expression is absent from C1 through C6 in the two year old bowhead whale (10B16, Fig. 30). These joints are fusing. The C6C7 intervertebral disc is unfused and BMP2/4 protein expression is present in endplate chondrocytes (Fig. 30). At 5-10 years of age (09B9), BMP2/4 protein expression is present in fewer joints, specifically it is absent in C1 through C4.

BMP2/4 is present in the cytoplasm of endplate chondrocytes of partially fused joints C5 through C7 (Fig. 30). There is an increase in BMP2/4 protein expression at 20 years old (10B15); BMP2/4 is present in all the joints in the endplate chondrocytes regardless of the amount of intervertebral fusion present.

Skeletonized Specimen and CT Scans

From the six of skeletonized bowhead cervical samples (09B11, 10B15,

10B16, 11B3, 11B8, 11B9) it was observed that intervertebral fusion begins at the center of the intervertebral disc and progresses peripherally (Fig. 31). The amount of intervertebral fusion increases ontogenetically (Fig. 31). The area of bony intervertebral fusion concurrently crosses the intervertebral space and the epiphyses, until all seven cervical vertebrae are fused (Fig. 31).

The CT scans of the one year old bowhead whale (09B11) confirm that intervertebral cervical fusion progresses from the center of the intervertebral joint,

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A two year old (10B16), a 5-10 year old (09B9), and a 20 year old (10B15) bowhead whale. Key for the fusion map in Figure 16. Red X- no expression, F- Fused joint, black X- missing sample, ep- endplate, b- bone. Sagittal sections at midline, 40x Brown represents positive staining for BMP2/4.

Figure 30. BMP2/4 protein expression in bowhead cervical intervertebral discs

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A- one year old (09B11), B- 2 year old (10B16), C- 5-10 year old (11B8), D- 20 year old (10B15), E- 20 year old (11B9), F- 40 year old (11B3). Notice and median and lateral views. Cranial to the left. Scale for A-E= 1 cm, scale for F = 1 cm.

Figure 31. Skeletonized bowhead whale ontogenetic cervical series

112 near the location where the nucleus pulposus was present, and progresses towards the periphery of the intervertebral joint (Fig. 32).

This ontogenetic skeletonized series of bowhead whales illustrates nicely the shortening of the cervical vertebrae in the cranial caudal dimension (Fig. 31).

In the 40 year old bowhead whale with all seven cervical vertebrae fused, the neck is only about one foot in length while the entire body length is 40 feet (Fig.

33). The foreshortening of cervical vertebrae is already present in a fetal bowhead whale (Fig. 34). In this specimen (00B3), Carnegie stage 23, the cervical vertebrae are much thinner and shorter in the cranial-caudal dimension than thoracic or post-thoracic vertebrae (Fig. 34).

Discussion

Identification of cervical intervertebral fusion pattern in the bowhead whale was established using protein expression (Figs. 27-30), skeletonized specimens

(Fig. 31), and CT scans (Fig. 32). In bowhead whales, cervical vertebral fusion begins at the center of the intervertebral disc near the nucleus pulposus and progresses peripherally towards the annulus fibrosus (Fig. 31 and 32).

The cervical intervertebral fusion pattern exhibited in the bowhead whale is different from that exhibited in the mouse, a terrestrial mammal (Fig. 35).

Sacral intervertebral fusion begins at the periphery of the intervertebral joint and progresses centrally, so the last region of the intervertebral disc to ossify is the nucleus pulposus (Fig. 35). The advantages of fusing vertebrae from the inside

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Cervical vertebrae 1-5 of one year old bowhead whale (09B11). Cranial to the left. Slices every .12mm. Notice the large region of intervertebral fusion occurring at midline and crossing intervertebral and epiphyseal cartilaginous regions.

Figure 32. CT scans of bowhead neck

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A- black and white line drawing of a 40 year old bowhead whale, B- neck of a 40 year old bowhead whale (11B3). Notice all seven cervical vertebrae are present in B and they total 33 cm in the cranial- caudal dimension compared to the body length 40 ft (1219 cm).

Figure 33. Bowhead body length compared to neck length

Alizarin red and alcian blue whole mount staining of a bowhead fetus Carnegie stage 23 (00B3). A- entire skeleton, B- close up of foreshortened neck, C- black and white line drawing of seven cervical vertebrae. Notice C1-4 are already fused in the neural arch. Red- bone, blue- cartilage.

Figure 34. Cleared and stained bowhead fetus

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A- one year old bowhead (09B11) cervical vertebrae, B- black and white line drawing of 09B11, C- 30 day old mouse (M78b) histological section, D- black and white line drawing of M78b. The gray shading is mineralized tissue, showing intervertebral fusion in whales begins centrally and leaves outer annulus fibrosus last, while mice mineralize the annulus fibrosus first and the nucleus pulposus last. Af- annulus fibrosus, C- cervical vertebrae 1-5, gp- growth plate, np- nucleus pulposus, S- sacral vertebrae 1-4, Notice the different intervertebral fusion patterns.

Figure 35. Intervertebral fusion patterns of bowhead whale and mouse

116 of the joint to the periphery of the joint or vice versa are currently unknown. The difference may be associated with terrestrial or aquatic locomotion and associated distributed forces. Archaic cetaceans are terrestrial locomotors and share the intervertebral fusion pattern exhibited in mice, therefore the sacrum may have only one pattern of intervertebral fusion regardless of species. During the evolution of cetaceans and the evolution of fully aquatic locomotion, this fusion pattern was pressured to change.

Sacral Intervertebral Fusion Patterns compared to Cervical Intervertebral Fusion

From the immunohistochemistry results, there is an ontogenetic maintenance of MMP13 and TIE2 protein expression, which differs from the pattern exhibited in the mouse sacrum; mice sacra exhibit an ontogenetic decrease of MMP13 and TIE2 (Table 2). GDF5 protein expression ontogenetically increases its presence in the number of joints expressing GDF5; this increase does not support the hypothesis and is not consistent with the mouse immunohistochemistry results (Table 2). BMP2/4 protein expression also ontogenetically increases expression in the number of joints with expression, which supports the hypothesis and agrees with the mouse results.

The only intervertebral disc region to exhibit protein expression was the endplates; very few cells were present in the annulus fibrosus and those that were present are fibroplastic in phenotype. The annulus fibrosus is dominated by collagen fibers. The nucleus pulposus was absent in all samples analyzed for

117 this project, further supporting the nucleus pulposus is the first region in the bowhead whale cervical intervertebral disc to fuse.

Table 2. Mouse and Bowhead Immunohistochemistry Results

Comparing MMP13, TIE2, GDF5, and BMP2/4 expression in the annulus fibrosus and the endplate of the intervertebral disc. The overall expression patterns mostly match the original predictions in mice, but the bowhead protein expression does not match the predictions and may be due to the ages of individuals.

The immunohistochemistry is not consistent between the mouse and bowhead whale. One reason may be that cetaceans tend to be delayed in growth and have long lifespan when compared to most mammals (Wheeler,

1930; Ohsumi et al., 1958; Kato, 1988; Ito and Miyazaki, 1990; Yoshida et al.,

1994; Kemper and Leppard, 1999; Best and Lockyer, 2002; Galatius, 2010).

Protein expression may be delayed in bowhead whales due to their long life spans and this may suggest that the ages of the bowhead whales used in this study are insufficient in identifying protein expression pattern.

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The ossification pattern of intervertebral fusion also differs between mice and bowhead whales. Intervertebral fusion in the mouse occurs from the periphery of the joint and ossification continues towards the nucleus pulposus near the center of the disc (Fig. 35). The bowhead whale intervertebral disc ossifies from the center, close to the nucleus pulposus, and progresses peripherally (Fig. 35). The reason for two ossification patterns in mammals is unknown, but may be associated with environment and locomotor pattern.

Functions of a Fused Neck

The functional significance for cervical intervertebral fusion is unknown. It could be that cervical intervertebral fusion occurs in response to maintaining the position of the large head in a streamlined orientation (Buchholtz, 2001). The cervical vertebrae of cetaceans are also unique because they are foreshortened in the cranial-caudal dimension (Buccholtz,1998). Shortening the neck of modern cetacean stiffens the neck (Fig. 9) and enhances the hydrodynamic shape of the body, and stabilizes the head and torso during fluke or tail propelled locomotion (Buchholtz, 1998; Fish, 2002; Fish et al., 2003). Together these cervical adaptations in cetaceans provide a skeleton designed for more efficient swimming.

119 orientation and movement (Spoor, 2003). Semicircular canal diameter is scaled allometrically with body size in terrestrial (Spoor, 2003) taxa such large bodied animals typically have large semicircular canals. Surprisingly, modern cetaceans have tiny semicircular canals relative to body size (Ketten, 1992; Spoor et al.,

2002). These reduced canals result in less sensitive balance, which in turn is closely related to sinusoidal head rotation and movement (Yang and Hullar,

2007). I was not able to identify any relationship between neck fusion and semicircular canal size (Chapter 2, Fig. 25).

Aging Bowhead Whales using Cervical Fusion

Bowhead whales are aged by the Department of Wildlife Management to ensure population maintenance. To age these whales, a multitude of techniques are used including body length, retinal analysis, and baleen length. As another technique to add to this list of aging techniques, cervical fusion was a good candidate as it changes ontogenetically (Fig. 16). However, from the seven individuals I sampled, the two twenty year old bowhead whales exhibit very different fusion patterns. Therefore too much variation exists in the amount and progression of cervical fusion to make this an accurate aging technique for bowhead whales.

Conclusion

The sacrum of terrestrial mammals and the neck of some cetaceans are two regions of normally occurring intervertebral fusion in the axial skeletons of

120 mammals. Here, the immunohistochemistry of both vertebral regions were compared t to determine if intervertebral fusion molecular mechanisms remain the same across mammalian species regardless of vertebral level.

Immunohistochemistry for ontogenetic bowhead cervical intervertebral fusion were inconsistent with the mouse results. The inconsistency may be due to the long lifespan of bowhead whales and the association delay in some biological processes. It may be possible similarities exist between the protein expression identified in the mouse sacrum and those of the bowhead whale and there is a need for bowhead whales older than 40 years of age.

The patterns of intervertebral fusion also differ between the mouse and the bowhead. In mice, intervertebral fusion begins peripherally in the intervertebral disc and progresses peripherally; the nucleus pulposus is the last region of the disc to ossify. In the bowhead whale, intervertebral fusion starts centrally and progresses peripherally suggesting that the nucleus pulposus is the first region of the disc to undergo ossification and bony fusion progresses from the inside of the disc towards the periphery.

There is also variation in the amount of intervertebral fusion in bowhead whales and for this reason, cervical intervertebral fusion of the bowhead whale is not a consistent aging technique for this species.

CHAPTER V

IDENTIFICATION OF THE SACRUM IN MODERN CETACEANS

Introduction

A critical problem in modern cetaceans is establishing the vertebral homologies of terrestrial mammals due to a lack of morphological indicators

(Slijper, 1936; Buchholtz, 1998; Buchholtz, 2010). Without intervertebral fusion to demarcate the sacrum in modern cetaceans two criteria are used to identify post-thoracic modern cetacean vertebrae. The first is based on the location of the first hemal arch or chevron bone (Eschricht et al., 1866; Slijper, 1936;

DeSmet, 1977). Hemal arches are V-shaped bones that articulate with the ventral side of the caudal vertebrae near the intervertebral joint and protect the aorta (DeSmet, 1977). The first hemal arch is considered to be located at the anterior (Eschricht et al., 1866; Slijper, 1936) or posterior (Wheeler, 1930;

DeSmet, 1977) border of the first caudal vertebra.

The second criterion for sacral identification in modern cetaceans is based on the location of the pudendal nerve as it exits the vertebral canal, a foramen formed when two vertebrae are articulated. The first root of the pudendal nerve exits the vertebral column caudal to the first sacral vertebra (Slijper, 1936).

Identification of the cetacean sacrum is of interest because modern cetaceans 121 122 present unique vertebral column morphology among mammals and as derived body types, we were curious if any sacral signaling was retained evolutionarily in the sacrum of modern cetaceans.

Therefore we identified developing sacra in two fetal cetaceans, Stenella attenuata (pantropical spotted dolphin) and Balaena mysticetus (bowhead whale), based on the location of the first root of the pudendal nerve relative to the developing vertebrae.

Materials and Methods

An ontogenetic series of fetal Stenella attenuata (n= 2) and one fetal

Balaena mysticetus (n= 1) were cleared and stained using the same technique used on the mouse series. Staging of the fetuses is based on the Carnegie system adapted for Stenella attenuata by Thewissen and Heyning (2007) and fetal age is based on Stěrba et al., (2000). The stained fetal specimens were late fetal stages from Carnegie stage 20-23. Skulls of the Stenella specimens were previously described in Moran et al. (2011). These fetal Stenella specimens are a museum collection from the Natural History Museum of Los Angeles County,

Los Angeles, CA.

Pudendal nerve dissections were completed in two late fetal (C23) cetacean specimens; Stenella attenuata (LACM 94310, n= 1) and Balaena mysticetus (00B3, n= 1). These dissections were completed to identify the anterior most of the developing sacral vertebra. This fetal bowhead specimen is

123 covered under the NOAA-NMFS 814-1899-01 permit with permission from the

Alaska Eskimo Whaling Commission and local subsistence hunters.

Results

We chose to pursue the pudendal nerve, a soft tissue landmark, to identify the sacrum in modern cetaceans as discussed by Slijper (1936). Pudendal nerve dissections were completed before the Stenella attenuata fetus, Carnegie stage

23 (Fig. 36A and B) and the Balaena mysticetus fetus, Carnegie stage 23 (Fig.

36C and D) were cleared and stained. The vertebral level where the first root of the pudendal nerve exited the vertebral column was recorded. In Stenella, the first root of the pudendal nerve exited posterior to the tenth lumbar (L10- Fig.

36B). In Balaena, the first root of the pudendal nerve exited at a different vertebral level. It exited at the seventh lumbar (L7- Fig. 36D).

Clearing and staining these cetacean fetuses exposed more densely ossified spinous processes in the sacral vertebrae (Fig. 37A and B). LACM

94310 (Fig. 37A) is a Stenella fetus from Carnegie stage 21/22 and has a total of

79 vertebrae with a vertebral formula of C7, T16, L11, S5, Ca40 (Fig. 37A).

Sixty-nine of these vertebrae are ossified and the last 10 caudal vertebrae are cartilaginous. There are five larger spinous process ossifications just anterior to the pelvis in post-thoracic vertebrae 13-17 (Fig. 37A). These ossifications are visible as dark triangles and are more ossified than adjacent vertebrae. The most cranial ossification is the smallest and just starting to ossify. These larger

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A, Stenella attenuata fetus, stage C21/22 (LACM 94268); B, black and white line drawing of S. attenuata pudendal nerve; C, Balaena mysticetus fetus, stage 23 (00B3); D, black and white line drawing of B. mysticetus pudendal nerve. The root of the pudendal nerve exits the vertebral canal caudal to the first sacral vertebra. cc, crus of corpus cavernosum; dn, dorsal nerve of the penis; ep, epididymus; fe, femur; ir, inferior rectal nerve; il, ilium; pe, penis; pn, pudendal nerve; ri, right innominate; S1, first sacral vertebra; sp, spinous process; te, testis; ti, tibia; tp, transverse, process; vr, ventral ramus.

Figure 36. Pudendal nerve of modern cetaceans

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A, S. attenuata, Carnegie stage C21/22 (LACM 94310); B, S. attenuata, stage C23 (LACM 94285); C, B. mysticetus, stage 23 (00B3). Notice the black triangular ossifications (bracketed) in the developing sacrum (A). These ossifications suggest an accelerated rate of ossification in the sacral region compared to the adjacent vertebrae (B, C).

Figure 37. Cleared and stained cetacean fetuses

126 sacral ossifications are also visible in an older Stenella fetus from Carnegie stage

23 (Fig. 37B). This fetus also has 79 total vertebrae and the same vertebral formula. The larger sacral ossifications are present in post-thoracic vertebrae

15-18; slightly more posteriorly located than in LACM 94310 (Fig. 37B). In LACM

94285, most of the spinous processes are ossified, but the sacral spinous process ossifications are larger than those of adjacent lumbar and caudal vertebrae (Fig. 37B). The larger spinous process ossifications correspond to the location of the sacrum as identified by the pudendal nerve dissection.

The Balaena mysticetus fetus (00B3) also exhibits larger vertebral ossifications, but they are located in the lamina and pedicle of the vertebra, not the spinous process as in Stenella (Fig. 37C). This Carnegie stage 23 bowhead has a total of 55 vertebrae, with a vertebral formula of C7, T13, L7, S4, Ca24.

The lamina and pedicle of the vertebrae are ossified for most of the column, but larger ossifications are present at post-thoracic vertebrae 3-7 (Fig. 37C). Unlike in Stenella, the larger vertebral ossifications exhibited in this bowhead do not correspond to the location of the sacrum as identified by pudendal nerve dissection.

Conclusion

The pudendal nerve has been used as a soft tissue landmark in identification of the sacrum. In this report, we show there is variation across species in the vertebral level where the pudendal nerve exits the vertebral

127 column and therefore other criteria need to be used when trying to identify sacral vertebra in modern cetaceans.

We also cleared and stained whole mount Stenella and Balaena specimens. Observations on the vertebral column yielded an interesting pattern of ossification in the sacral region. Larger ossifications are present in the spinous processes, lamina and pedicle of the sacral vertebrae in these two species of modern cetaceans. This suggests that, even though the sacrum of modern cetaceans never fuses, some molecular signaling is retained causing accelerated ossification in the sacral vertebrae compared to adjacent vertebral regions.

CHAPTER VI

A REVIEW OF INTERVERTEBRAL AND EPIPHYSEAL FUSION IN CETACEANS AND TERRESTRIAL MAMMALS

In this study, I focused on the variation of patterns and mechanisms of intervertebral and epiphyseal fusion in archaic and extant cetacean taxa and extant terrestrial mammals. Further understanding of mammalian morphological and molecular variation and expansion of scientific understanding of the relationship between phenotype and genotype (Willmore et al., 2007) is the heart of this project. I utilized a variety of techniques from computerize tomography

(CT) and gross dissection to histology and immunohistochemistry to pursue the morphological, paleontological, molecular, and developmental variations in the patterns of intervertebral and epiphyseal fusion in the vertebral column.

The main hypothesis stated genes expressed during endochondral ossification are expressed during intervertebral fusion. To support this hypothesis four aims were formulated.

1) To determine if endochondral ossification of long bones is a model for

intervertebral fusion, specific molecules of interest were analyzed

including MMP13, TIE2, COLX, and BMP2/4). Other molecules studied

include ANK, GDF5, and GDF6.

128 129

2) To identify the ontogenetic patterns of intervertebral and epiphyseal fusion

within the vertebral column of terrestrial and aquatic mammals to further

understand mammalian variation in an evolutionary context.

3) To determine the ontogenetic intervertebral fusion patterns in the cervical

intervertebral discs of bowhead whales (Balaena mysticetus)

4) To determine if the patterns of protein expression exhibited in sacrum of

terrestrial mammals (mouse) are the same as in the neck of some modern

cetaceans (bowhead whale) in order to identify

This study is unique from other vertebral or intervertebral disc research because it focuses on naturally occurring fusion models- the sacrum of land mammals and the necks of bowhead whales. These vertebral regions are preprogrammed for intervertebral fusion to occur and if we as scientists can understand the mechanisms of intervertebral fusion in a normal model, which may include bone formation and endochondral ossification, then this knowledge can be applied to translational research and clinical applications.

This project yielded three main implications: 1) Evolutionary implications,

2) Conservation Management implications, and 3) Biomedical implications.

These three themes provide a setting for the importance of this study.

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Evolutionary Implications

Evolutionary implications were drawn from the analyses on extinct and extant cetaceans and extant terrestrial mammals (Chapters 2 and 3). The pattern of intervertebral fusion within a single vertebral joint is also evolutionarily significant due to the variation present across mammals (Chapters 2 and 3). In mice and pigs intervertebral fusion occurs from the periphery of the intervertebral disc and progresses centrally (Fig. 5 and 14). Archaic cetaceans, Ambulocetus and Kutchicetus, exhibit the same intervertebral fusion pattern as the modern terrestrial mammals (Fig. 15). The pattern of sacral intervertebral fusion is the same in mice and pigs (Fig. 14) as is Ambulocetus natans and Kutchicetus minimus (Fig. 15). The number of fused sacral vertebrae changes evolutionarily and is associated with locomotor style and environment (Fig. 23). Sacral formation and sacral intervertebral fusion progresses in a cranial to caudal pattern in terrestrial mammals. Our results confirm sacral intervertebral fusion pattern is retained evolutionarily (Fig. 14 and 15).

The sacrum in cetaceans is a good example of skeletal variation present in mammals. Throughout whale evolution, the number of fused sacral vertebrae decreases from four fused sacral vertebrae to none in their modern cetacean descendants (Fig. 22). Identification of post-thoracic vertebrae in modern cetaceans is difficult due to the lack of bony landmarks and regionalization in the vertebral column (Chapter 5). Though modern cetaceans exhibit homologous morphology in post-thoracic vertebrae, I identified modern cetaceans retain

131 developmental signals in the sacral region that cause these vertebrae to ossify earlier than adjacent vertebrae (Fig. 23). The function of this accelerated ossification is unknown, but may be associated with the dorsoventral undulation characteristic of cetacean swimming.

Intervertebral fusion is not lost completely in some modern cetaceans. In the bowhead whale all seven cervical vertebrae fuse together (Eschricht et al.,

1866; Wheeler, 1930; Slijper, 1936; Van Valen, 1967; Haldiman and Tarpley,

1993; Buchholtz, 2001; Buchholtz et al., 2005; Buchholtz, 2007; Buchholtz et al.,

2007). Intervertebral fusion in the cervical vertebrae exhibit a very different pattern of fusion from the terrestrial mammals. Cervical intervertebral fusion in the bowhead whale begins at the center of the intervertebral disc and progresses peripherally (Fig. 16, 31, and 32). The number of fused cervical vertebrae varies across species (Fig. 24) and little relationship was identified when cervical intervertebral fusion was interpreted with semicircular canal size (Fig. 25).

Evolutionary changes are present in epiphyseal fusion patterns as well. I found epiphyseal fusion patterns differ between terrestrial and aquatic mammals.

In terrestrial mammals, epiphyseal fusion begins in the cervical and sacral vertebral regions simultaneously and both fusion progresses caudally (Figs.17 and 18). The last epiphyses to fuse are in the lower thoracic region.

Ambulocetus natans exhibits an epiphyseal fusion pattern similar to modern terrestrial mammals (Figs. 19 and 38), while the epiphyseal fusion pattern of

Kutchicetus minimus is like modern cetaceans (Fig. 19 and 38). Epiphyseal

132

Epiphyseal fusion in modern terrestrial, archaic, and modern aquatic mammals

Figure 38. Summary of epiphyseal fusion

133 fusion in Kutchicetus and modern bowhead and beluga whales begins in the cervical and caudal vertebra first and epiphyseal fusion progresses towards mid- column (Figs. 19 and 38).

Conservation Management and Aging in Cetaceans

Aging bowhead whales is part of the extensive conservation management in place to help sustain pod sizes, reproduction, and overall survival of the species. Two fusion patterns exhibited in the vertebral column can be applied to aging cetaceans, specifically, bowhead whales. I have shown that epiphyseal and intervertebral fusion patterns change ontogenetically. In modern cetaceans, epiphyseal fusion starts in the cervical and caudal vertebrae simultaneously and progress towards the middle of the vertebral column. Intervertebral fusion in modern cetacean species exhibits a pattern of fusion that begins near the nucleus pulposus at the center of the intervertebral disc and progresses peripherally leaving the annulus fibrosus to be the last to ossify. Epiphyseal fusion patterns are already used to age many species of cetaceans (Wheeler,

1930; Kato, 1988; Ito & Miyazaki, 1990; Mead & Potter, 1990; Yoshida et al.,

1994; Kemper & Leppard, 1999; Best & Lockyer, 2002; Galatius, 2010).

Epiphyseal fusion can be used to age bowhead whales as well though the process is laborious and not time efficient. To alleviate some of the logistical difficulties, I thought cervical fusion could be used as an alternative to epiphyseal fusion to age bowhead whales. However, there is too much variation in the

134 amount of intervertebral fusion in the cervical vertebrae of the bowhead whale to make this technique one that is useful (Fig. 31).

Biomedical Application of Research

In this project, I focused on regions of intervertebral fusion that occur during normal ontogeny. If the mechanisms of normal non-pathological fusion can be identified and understood then this information can potentially be applied to bone formation and joint fusion treatments. Understanding bone formation can help reduce bone when needed as in bone spurs or increase bone formation as in spinal fusions and fracture healing.

I chose endochondral ossification as a model for intervertebral fusion and pursued patterns of protein expression associated with endochondral ossification in the intervertebral discs of the sacrum of mice. Results yielded more similarities between non-fusing and fusing joints than expected, but this is only the first step in this research and there are other molecules of interest that can be pursued. Perhaps endochondral ossification is only occurring in part of the intervertebral disc like the annulus fibrous and the endplates. The nucleus pulposus is retained long past when the intervertebral joint is considered fused and has lost mobility.

Two Fusion Processes and the Big Picture

In this dissertation, I investigated intervertebral fusion and epiphyseal fusion in two regions of normal intervertebral fusion to further understanding of

135 mammalian variation throughout evolution (Chapters 2-4). I showed there are two distinct intervertebral fusion patterns present in terrestrial and aquatic mammals. Sacral intervertebral fusion during sacral formation begins in the annulus fibrosus, the outermost region of the intervertebral disc, and progresses centrally (Chapter 3). This is different from cervical intervertebral fusion in the bowhead whale; intervertebral fusion begins in the nucleus pulposus and progresses peripherally (Chapter 3).

These two types of fusion processes are considered to be controlled by separate signaling pathways, but this may not be the case. There may be some relationship between intervertebral and epiphyseal fusion because I showed that epiphyseal fusion starts in the same locations where intervertebral fusion is present (the sacrum and the neck, Figs. 17-20, 38).

Also in attempt to identify a molecular fusion module, I compared protein expression in the sacral intervertebral disc of a terrestrial mammal model, the mouse, to the cervical intervertebral fusion in an aquatic mammal, bowhead whales. I am able to show protein expression of three of our seven molecules exhibit different expression patterns between non-fusing and fusing sacral joints of the mouse (Chapters 2 and 4).

Intervertebral fusion may be controlled by endochondral ossification during which the breakdown of extracellular matrix occurs in order for bone to form linking vertebrae together. There seems to be many possibilities for change in this region and more research needs to be executed. Conversely, epiphyseal

136 fusion is also dictated by endochondral ossification but strictly controlled by the

PTHrP-Ihh loop, which is responsible for bone growth. If this pathway is interrupted, by plasticity or otherwise, the repercussions impact life of the individual.

Future Research

The first future research project will be real time reverse transcriptase

PCR; this will be completed in intervertebral discs of fusing regions, including the mouse sacrum and the cervical region of the bowhead whale. Some of the genes of interest are evolutionarily conserved, such as BMP2, BMP4, and GDF5

(Leong and Brickell, 1996). Due to the homologous nature of BMPs I expect consistent results in the whale samples through the commercially made antibodies are not specific for cetaceans. This research will strengthen the patterns of gene expression in fusing intervertebral joints already established by immunolocalization research.

Other future research includes the study of knockout mice. Knocking out some the genes of interest result in embryonic lethality (BMP2- Ducy and

Karsenty 2000; BMP4- Ducy and Karsenty 2000; Goldman et al 2009; TIE2-

Yancopolous et al. 2000; Eklund and Olsen 2006; Makinde and Agrawal 2008).

GDF5, ANK, MMP13, and type X collagen have viable knockout phenotypes. A potential project under discussion is a knockout (KO) study on type X collagen null mice, in collaboration with Olena Jacenko’s lab at UPenn. This project aims

137 to identify how sacral fusion is affected by the lack of collagen X compared to wild type mice.

This project focuses on mammalian variation and the basis of this project can be expanded to include a variety of other model species, like the jerboa.

Other techniques can be utilized like organ culture of mouse sacra. Organ culture could be pursued in order to further support the roles of molecules associated with endochondral ossification in an in vitro experiment. Molecules of interest can be expanded to include Runx2 or Sox9. Both bone formation transcription factors present upstream from those molecules of interest pursued in this project. Ultimately, this project opens up multiple avenues of research to continue down evolutionary, morphological, molecular, and developmental paths to interesting and fulfilling science.

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