The ancient gene c12orf29 an exploration of its role in the chordate body plan

THE ANCIENT GENE C12ORF29:
AN EXPLORATION OF ITS ROLE IN THE
CHORDATE BODY PLAN

Thor Einar Friis
Bachelor of Science (Hons)

Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy

Institute of Health and Biomedical Innovation
Faculty of Built Environment and Engineering
Queensland University of Technology
January 2013

 


 




 

Keywords 


Complementary DNA library



Evolutionary biology



Developmental biology



Skeletal development



Cartilage



Phylogeny



Chordate body plan

 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 

i 

 

 

ii 

 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 


 

Abstract
The sheep (Ovis aries) is commonly used as a large animal model in skeletal research. Although the sheep genome has been sequenced there are still only a limited
number of annotated mRNA sequences in public databases. A complementary DNA
(cDNA) library was constructed to provide a generic resource for further exploration
of genes that are actively expressed in bone cells in sheep. It was anticipated that the
cDNA library would provide molecular tools for further research into the process of
fracture repair and bone homeostasis, and add to the existing body of knowledge.
One of the hallmarks of cDNA libraries has been the identification of novel genes
and in this library the full open reading frame of the gene C12orf29 was cloned and
characterised. This gene codes for a protein of unknown function with a molecular
weight of 37 kDa. A literature search showed that no previous studies had been conducted into the biological role of C12orf29, except for some bioinformatics studies
that suggested a possible link with cancer. Phylogenetic analyses revealed that
C12orf29 had an ancient pedigree with a homologous gene found in some bacterial
taxa. This implied that the gene was present in the last common eukaryotic ancestor,
thought to have existed more than 2 billion years ago. This notion was further supported by the fact that the gene is found in taxa belonging to the two major eukaryotic branches, bikonts and unikonts. In the bikont supergroup a C12orf29-like
gene was found in the single celled protist Naegleria gruberi, whereas in the unikont
supergroup, encompassing the metazoa, the gene is universal to all chordate and,

therefore, vertebrate species. It appears to have been lost to the majority of cnidaria
and protostomes taxa; however, C12orf29-like genes have been found in the cnidarian freshwater hydra and the protostome Pacific oyster. The experimental data
indicate that C12orf29 has a structural role in skeletal development and tissue homeostasis, whereas in silico analysis of the human C12orf29 promoter region suggests that its expression is potentially under the control of the NOTCH, WNT and
TGF- developmental pathways, as well SOX9 and BAPX1; pathways that are all
heavily involved in skeletogenesis. Taken together, this investigation provides strong
evidence that C12orf29 has a very important role in the chordate body plan, in early
skeletal development, cartilage homeostasis, and also a possible link with spina bifida in humans.
The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 

iii 


 

iv 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 


 

Table of Contents 
Keywords .................................................................................................................................................. i 
Abstract .................................................................................................................................................. iii 
Table of Contents .................................................................................................................................... v 
List of Figures ....................................................................................................................................... viii 
List of Tables .......................................................................................................................................... xii 
List of Abbreviations ............................................................................................................................. xiii 
Statement of Original Authorship ....................................................................................................... xxii 
Acknowledgements ............................................................................................................................ xxiii 

CHAPTER 1:  INTRODUCTION ...................................................................................................... 1 
1.1 

Background .................................................................................................................................. 1 

1.2 

Context ......................................................................................................................................... 2 

1.3 

Purposes ....................................................................................................................................... 3 

1.4 

Thesis Outline .............................................................................................................................. 4 

CHAPTER 2:  LITERATURE REVIEW .............................................................................................. 7 
2.1 

A brief introduction to skeletal biology ....................................................................................... 7 

2.2 

Complementary DNA libraries and their utility in biological research...................................... 32 

2.3 

The sheep as an animal model ................................................................................................... 35 

2.4 

Summary and Implications ........................................................................................................ 37 

CHAPTER 3:  METHODS AND MATERIALS .................................................................................. 41 
3.1 

Methodology and Research Design ........................................................................................... 41 
3.1.1  Introduction .................................................................................................................... 41 

3.2 

Construction of a cDNA library to identify genes expressed in cells derived from sheep bone 44 
3.2.1  Cell culture ...................................................................................................................... 44 
3.2.2  RNA extraction ................................................................................................................ 45 
3.2.3  cDNA library construction ............................................................................................... 47 
3.2.4  PCR based methods for isolating full length cDNA clones .............................................. 53 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 

v 


 

3.2.5  Library Screening: Method for amplification and isolation of cDNA from plasmid 
libraries that require no hybridization (MACH) .............................................................. 53 
3.2.6  Validation of cDNA library .............................................................................................. 57 
3.3 

C12orf29 – Characterization of a protein of unknown fuction .................................................. 59 
3.3.1  Subcloning C12orf29 cDNA into an epitope tagged expression vector .......................... 60 
3.3.2  Validation of C12orf29 antibody specificity by western blot (WB) analysis ................... 75 
3.3.3  Immunofluorescent (IF) microscopy ............................................................................... 83 
3.3.4  Immunohistochemistry (IHC) .......................................................................................... 85 
3.3.5  RT‐qPCR analysis of C12orf29 expression in sheep primary cells ................................... 88 
3.3.6  WB analysis of C12orf29 protein expression in sheep primary cells .............................. 90 

3.4 

Bioinformatics analyses of C12orf29 ......................................................................................... 91 
3.4.1  Phylogenetic analyses ..................................................................................................... 91 
3.4.2  Molecular genetics analyses ........................................................................................... 94 

CHAPTER 4:  RESULTS .............................................................................................................. 99 
4.1 

Results – Introduction ................................................................................................................ 99 

4.2 

cDNA library construction ........................................................................................................ 100 
4.2.1  Isolation of GAPDH cDNA clone using the MACH protocol .......................................... 107 
4.2.2  Validation of the cDNA library ...................................................................................... 111 

4.3 

C12orf29 – Experimental Results ............................................................................................. 117 
4.3.1  Subcloning and epitope tagging the C12orf29 cDNA clone .......................................... 117 
4.3.2  Validating the specificity of the anti‐C12orf29 antibodies ........................................... 119 

4.3.3  Immunofluorescent microscopy ................................................................................... 125 
4.3.4  Immunohistochemistry ................................................................................................ 130 
4.3.5  Real time quantitative PCR analysis ............................................................................. 145 
4.3.6  WB analysis of C12orf29 protein expression in sheep primary cells ............................ 147 

4.4 

C12orf29 – Bioinformatics analyses ......................................................................................... 149 
4.4.1  Phylogenetic analyses ................................................................................................... 149 
4.4.2  Molecular genetics analyses ......................................................................................... 156 

CHAPTER 5:  ANALYSIS AND DISCUSSION ............................................................................... 173 
5.1 
vi 

Introduction ............................................................................................................................. 173 
The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 


 

5.2 

The cDNA libary – Analysis and discussion .............................................................................. 174 
5.2.1  A brief review of some of the genes isolated from the cDNA library ........................... 176 
5.2.2  cDNA library construction – Conclusions ...................................................................... 200 

5.3 

Experimental characterization of C12orf29 – Analysis and Discussion ................................... 206 

5.3.1  Molecular cloning experiments .................................................................................... 206 
5.3.2  Validation of the commercial C12orf29 antibodies ...................................................... 207 
5.3.3  Immunofluorescent microscopy – Discussion .............................................................. 211 
5.3.4  Immunohistochemistry – Discussion ............................................................................ 212 
5.3.5  RT‐qPCR and western blot analysis – Discussion .......................................................... 217 

5.4 

Discussion – C12orf29 phylogenetic analyses .......................................................................... 219 
5.4.1  Distribution of C12orf29‐like proteins in eukaryata ..................................................... 221 
5.4.2  C12orf29 and the chordate superphylum .................................................................... 226 
5.4.3  Vertebrates – The Craniata ........................................................................................... 234 
5.4.4  Conclusions – Tracing C12orf29 through biological time and space ............................ 236 

5.5 

Discussion – Promoter Analysis ............................................................................................... 242 

CHAPTER 6:  CONCLUSIONS AND FUTURE WORK .................................................................... 247 
6.1 

Conclusions and future work – Introduction ........................................................................... 248 
6.1.1  The ancient gene C12orf29 – Putting together the pieces of the puzzle ..................... 249 

6.2 

C12orf29: the working hypothesis – What does it do? ............................................................ 254 

6.3 

Future work .............................................................................................................................. 256 

BIBLIOGRAPHY  .......................................................................................................................... 261 
APPENDICES   .......................................................................................................................... 299 
APPENDIX A – MICROBIOLOGY MATERIALS ........................................................................................ 300 
APPENDIX B – METHODS ..................................................................................................................... 301 
APPENDIX C – TRANSCRIPTS OF CDNA LIBRARY SEQUENCES SUBMITTED TO GENBANK ................... 323 
APPENDIX D – ALL KNOWN C12ORF29 PROTEIN SEQUENCES (DEC 2012) ......................................... 356 
APPENDIX E – STATISTICAL ANALYSIS ................................................................................................. 365 
APPENDIX F – TRANSCRIPT FROM THE SCIENCE SHOW ON ABC RADIO NATIONAL ........................... 366 
APPENDIX G – CONFERENCES AND PUBLICATIONS ............................................................................ 368 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 

vii 


 

List of Figures 
Figure 2‐1: Schematic cross‐section of the trunk of a vertebrate. .......................................................... 9 
Figure 2‐2: The somite forms two main components: sclerotome and dermomyotome. .................... 10 
Figure 2‐3: Ventral view of vertebral columns of Pax1 null and classical undulated 
homozygous mice at the newborn stage.  .................................................................... 15 
Figure 2‐4: Roentgenography of cervical vertebrae of a 25 year old man with KFS. ............................ 17 
Figure 2‐5: Schematic of the branchial arches and the development of the upper and lower 
jaws. ..................................................................................................................................... 20 
Figure 2‐6: Cells acquire their positional value in the progress zone that is specified at the 
distal end of the bud by the apical ectodermal ridge. ......................................................... 21 
Figure 2‐7: Establishing the DV axis of the limbs. ................................................................................. 23 

Figure 2‐8: Models of PD limb axis development. ................................................................................ 25 
Figure 2‐9  The BMP protein family. ..................................................................................................... 28 
Figure 2‐10: The BMP signalling cascade. ............................................................................................. 30 
Figure 2‐11: Constructing a cDNA library .............................................................................................. 34 
Figure 2‐12: WNT/‐Catenin, FGF, NOTCH, Hedgehog, and TGF/BMP are the major pathways 
regulating skeletal development. ......................................................................................... 37 
Figure 3‐1: Effect of in vitro Dex treatment of human BMSCs. ............................................................. 42 
Figure 3‐2: The sequence of the 50‐base oligonucleotide primer. ....................................................... 47 
Figure 3‐3: MACH library screening protocol. ....................................................................................... 55 
Figure 3‐4: The pcDNA3.1(+) expression vector.................................................................................... 62 
Figure 3‐5: The pcDNA3.1‐HA vector was double digested with EcoRV/XhoI....................................... 63 
Figure 3‐6: Pre‐ligation quality control. ................................................................................................ 64 
Figure 3‐7: An analytical digest of the plasmid preps ........................................................................... 67 
Figure 3‐8: Sequencing results of C12orf29 inserts. ............................................................................. 67 
Figure 3‐9: Strategy to retrofit an HA sequence into pcDNA‐C12orf29. ............................................... 69 
Figure 3‐10: Double digestion of the pcDNA3.1‐C12orf29 plasmid.. .................................................... 70 
Figure 3‐11: Nanodrop quantification of annealed HA oliogonucleotides. .......................................... 71 
Figure 3‐12: PCR analysis of colonies selected from plate #1 ............................................................... 73 
Figure 3‐13: Analytical digest of colonies identified as potential carriers of the HA tag. ..................... 74 
Figure 3‐14: WB analysis of C12orf29 expression with Abcam C12orf29 antibody. ............................. 76 
Figure 3‐15: WB analysis of C12orf29 expression with Santa Cruz C12orf29 antibody. ....................... 77 
Figure 3‐16: WB analysis of C12orf29 expression with Sigma C12orf29 antibody ............................... 78 
Figure 3‐17: WB analysis of C12orf29 expression with Everest Biotech C12orf29 antibody. ............... 79 
viii 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 


 

Figure 3‐18: In humans, the gene C12orf29 is located on the long arm of chromosome 12. ............... 94 
Figure 3‐19: Defining the cis‐regulatory region for the human C12orf29 gene by phylogenetic 
footprinting. ......................................................................................................................... 96 
Figure 4‐1: Shows the effect osteogenic induction medium on cell morphology. .............................. 100 
Figure 4‐2: Quantification of cDNA fractions on an ethidium bromide gel......................................... 102 
Figure 4‐3: Pilot ligation and transformation. ..................................................................................... 103 
Figure 4‐4: The analytical digests revealed the presence of inserts in all of the plasmids ................. 104 
Figure 4‐5: Three cDNA clones were isolated using the MACH protocol. ........................................... 107 
Figure 4‐6: The Ovis aries GAPDH protein sequence. ......................................................................... 108 
Figure 4‐7: Very highly expressed genes in the cDNA library. ............................................................. 111 
Figure 4‐8: Highly expressed genes in the cDNA library. .................................................................... 112 
Figure 4‐9: Lowly expressed genes in the cDNA library. ..................................................................... 112 
Figure 4‐10: Genes with the lowest expression in the cDNA library. .................................................. 113 
Figure 4‐11: The relative expression of all the genes tested on a logarithmic scale. .......................... 113 
Figure 4‐12: Effect of osteogenic induction medium on BMSCs, mOBs, and tOBs. ............................ 116 
Figure 4‐13: DNA and protein sequence of the recombinant HA‐C12orf29 plasmids. ....................... 118 
Figure 4‐14: Western blot experiment using the Licor double antibody labelling system. ................ 119 
Figure 4‐15: The Abcam anti‐C12orf29 antibody detects the HA‐C12orf29 recombinant protein ..... 120 
Figure 4‐16: Anti‐C12orf29 antibodies detect protein bands of the same molecular weight in 
3T3‐E1 cell lysates. ............................................................................................................. 121 
Figure 4‐17: Anti‐C12orf29 antibodies detect protein bands of the same molecular weight in 
C‐28/12 cell lysates. ........................................................................................................... 122 
Figure 4‐18:  EB49 and Santa Cruz anti‐C12orf29 antibodies were tested on CHO cell lysates 
overexpressing recombinant C12orf29. ............................................................................. 123 
Figure 4‐19: The Abcam and EB49 anti‐C12orf29 antibodies were tested on 3T3‐E1 cell 
lysates.. ............................................................................................................................... 124 
Figure 4‐20: Murine 3T3‐E1 cells transfected with HA‐C12orf29 plasmid and probed with an 
anti‐HA antibody. ............................................................................................................... 125 
Figure 4‐21: Sheep mandible osteoblast cells labelled with Abcam anti‐C12orf29 antibody and 
Phalloidin/DAPI.. ................................................................................................................ 126 

Figure 4‐22: Sheep PDL cells labelled with Abcam anti‐C12orf29 antibody and Phalloidin/DAPI. ..... 127 
Figure 4‐23: Sheep PDL cells. The C12orf29 protein appears to accumulate at the edges of 
many of the cells (arrowheads). ......................................................................................... 128 
Figure 4‐24: PC3 cells labelled with Abcam anti‐C12orf29 and Phalloidin/DAPI. ............................... 129 
Figure 4‐25: Normal human full thickness articular cartilage. ............................................................ 131 
Figure 4‐26: Osteoarthritic human articular cartilage. ....................................................................... 132 
Figure 4‐27: Rat tibial head showing the growth plate and trabecular bone. .................................... 133 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 

ix 


 

Figure 4‐28: Rat femoral head growth plate. ...................................................................................... 134 
Figure 4‐29: Mouse embryo at 13.5 dpc. ............................................................................................ 136 
Figure 4‐30: Human embryo sample at 7–8 weeks of gestation ........................................................ 137 
Figure 4‐31: COL2 expression in the developing human axial skeleton. ............................................. 138 
Figure 4‐32: C12orf29 expression in the developing axial skeleton of a human embryo at 7–8 
weeks gestation using the Abcam C12orf29 antibody. ...................................................... 139 
Figure 4‐33: C12orf29 expression in the developing axial skeleton of a human embryo at 7–8 
weeks gestation using the EB49 C12orf29 antibody. ......................................................... 141 
Figure 4‐34: SOX9 and BAPX1 expression in the developing human axial skeleton.. ......................... 143 
Figure 4‐35: The melt curve analysis for the sheep C12orf29 primers ............................................... 145 
Figure 4‐36: RT‐qPCR analysis showed there is clear effect of osteogenic induction medium on 
the gene expression of C12orf29 ....................................................................................... 146 
Figure 4‐37: WB analysis of cell lysates from sheep primary cells treated with osteogenic 
induction medium. ............................................................................................................. 147 
Figure 4‐38: Sequence alignment of human C12orf29 against orthologous proteins from the 

basal species identified by BLASTP search. ........................................................................ 152 
Figure 4‐39: Baysian phylogenetic reconstruction of C12orf29 using MrBayes. ................................ 154 
Figure 4‐40: In primates there is a cryptic exon (exon 1a) within the first intron. ............................. 157 
Figure 4‐41: The human C12orf29 gene has a distinct 500 bp CpG island.......................................... 160 
Figure 4‐42: Schematic showing the structural organisation of an RNA polymerase II 
promoter. ........................................................................................................................... 161 
Figure 4‐43: C12orf29 promoter sites identified by the TFSitescan search. ....................................... 163 
Figure 4‐44: Core promoter region of human C12orf29 showing a TATA box and binding sites 
for Sp1, c‐MYC, CSL and HES‐1. .......................................................................................... 166 
Figure 4‐45: SOX consensus binding sites in the promoter region of the human C12orf29 gene. ..... 168 
Figure 4‐46: NKX3.2 consensus binding sites in the C12orf29 promoter region. ............................... 169 
Figure 4‐47: TCF/LEF consensus DNA binding motifs in the C12orf29 promoter region. ................... 170 
Figure 4‐48: The DNA sequence motif CAGA is essential and sufficient for the induction of 
TGF‐ responsive genes. .................................................................................................... 171 
Figure 5‐1: The first reports of cDNA cloning and libraries appeared in 1975. ................................... 174 
Figure 5‐2: OPN and OCN mRNA expression is downregulated by 10‐7 M dexamethasone in 
sheep osteoblasts. .............................................................................................................. 202 
Figure 5‐3: An initial RT‐qPCR assay indicated that C12orf29 gene expression was upregulated 
in response to osteogenic induction medium. ................................................................... 206 
Figure 5‐4: Periodontal ligament cell stained with the Abcam C12orf29 antibody. ........................... 211 
Figure 5‐5: C12orf29 expression in tibial head in the rat. ................................................................... 212 
Figure 5‐6: Comparison of C12orf29 signal in the cranial vault of the forebrain in 13.5 dpc 
mouse embryos. ................................................................................................................. 213 

x 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 


 

Figure 5‐7: The H.chejuensis HCH_06039 protein contains a conserved domain with similarity 
to the adenylation DNA ligase superfamily.. ...................................................................... 220 
Figure 5‐8: The free‐living protist Nagleria’s predominant body form is amoeboid ........................... 221 
Figure 5‐9: The freshwater Hydra is a member of the Cnidaria superphylum, class medusozoa. ...... 222 
Figure 5‐10: The Pacific oyster Crassostera gigas. .............................................................................. 223 
Figure 5‐11: Amino acid alignment of human C12orf29 against Pacific oyster C12orf29‐like 
protein.. .............................................................................................................................. 224 
Figure 5‐12: Molecular phylogenetic analysis of C12orf29 by ML method using MEGA5. ................. 225 
Figure 5‐13: Schematic showing the principal common features of the Chordate superphylum. ..... 226 
Figure 5‐14: A simplified phylogenetic tree showing the relationship between Bilateria and 
Cnidaria. ............................................................................................................................. 227 
Figure 5‐15: A juvenile acorn worm at day 13 of development. ......................................................... 228 
Figure 5‐16: The acorn worm has two putative C12orf29 orthologs .................................................. 229 
Figure 5‐17: Ciona intestinalis larva. ................................................................................................... 230 
Figure 5‐18: The ascidian species C. intestinalis and C. savignyi each carry a copy of the 
C12orf29 gene.. .................................................................................................................. 231 
Figure 5‐19: The amphioxus Branchiostoma floridae ......................................................................... 232 
Figure 5‐20: Phylogenetic relationships of extant chordates. ............................................................. 234 
Figure 5‐21: A BLASTN search returned 138 lamprey contigs with sequence similarity to 
human C12orf29. ................................................................................................................ 235 
Figure 5‐22: Tracing C12orf29 through biological time and space. .................................................... 241 
Figure 6‐1: Protein alignment comparing human C12orf29 protein with the zebrafish C12orf29 
homolog. ............................................................................................................................ 256 
Figure 6‐2: C12orf29 microsynteny between zebrafish and humans. ................................................ 257 
Figure 6‐3: The promoter region of D. rerio C12orf29 gene. .............................................................. 258 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 

xi 

 

List of Tables 
Table 3‐1: M13 universal primer sequences. ........................................................................................ 51 
Table 3‐2: MACH primer sequences for sheep GAPDH ......................................................................... 54 
Table 3‐3: PCR cycling parameters used for the MACH isolation protocol. .......................................... 54 
Table 3‐4: List of primers used for RT‐qPCR validation of cDNA library................................................ 58 
Table 3‐5: The cloning primers used for subcloning C12orf29 into a pcDNA3.1(+) plasmid ................ 61 
Table 3‐6: Two reactions were setup to amplify the C12orf29 ORF with the cloning primer. ............. 61 
Table 3‐7: Touchdown temperature cycling conditions for C12orf29 PCR amplification. .................... 61 
Table 3‐8: Primer sequence of retrofit HA tag. ..................................................................................... 68 
Table 3‐9: Colony numbers from transformation with HA retrofitted plasmids. ................................. 72 
Table 3‐10: Colony numbers following HindIII digest of HA retrofitted plasmids ................................. 73 
Table 3‐11: DNA plasmid yields from colonies identified from analytical PCR reaction. ...................... 74 
Table 3‐12: Reagent volumes for transfection for antibody specificity experiments. .......................... 80 
Table 3‐13: Primary antibody solutions for WB analysis. ..................................................................... 82 
Table 3‐14: List of primers used for RT‐qPCR of C12orf29 mRNA expression in sheep cells ................ 89 
Table 4‐1: The total RNA yields and absorbance ratios produced with the RNA extraction kit. ........ 101 
Table 4‐2: Yield of poly(A)+ mRNA from the Nucleotrap Poly(A)+ enrichment kit. ............................. 102 
Table 4‐3: A list of all clones identified in the cDNA library. ............................................................... 105 
Table 4‐4: The H.chejuensis HCH_06039 protein, at 205 amino acids, is 2/3 the length of the 
eukaryotic C12orf29 homologs. ......................................................................................... 150 
Table 4‐5: Numerical evaluation of sequence similarities of C12orf29‐like proteins. ........................ 152 
Table 4‐6: Species comparison of intron/exon boundaries of the gene structure of C12orf29. ........ 158 
Table 4‐7: Compilation of putative transcription factor binding sites in the 5’ region of human 
C12orf29. ............................................................................................................................ 162 

xii 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 


 

List of Abbreviations 
A

 amyloid

ACAN

Aggrecan

ACTB

-actin

AD

Alzheimer’s disease

AER

Apical ectodermal ridge

ALDH

Aldehyde dehydrogenase

Amp

Ampicillin

ANTP

Antennapedia

AP

Anteroposterior

AP2

Activating Protein 2

APP

amyloid precursor protein

ARS

Alizarin red S

AXIN

Axis inhibition proteins

BA

Branchial arches

BAPX1

Bagpipe homeobox homolog 1

BAT

Brown adipose tissue

BGLAP

Bone gamma-carboxyglutamic acid protein

bHLHZip

Basic helix-loop-helix-leucine zipper

BLAST

Basic Local Alignment Search Tool

BMP7

Bone morphogenic protein 7

BMSCs

Bone marrow stromal cells

bp

Basepair

BSA

Bovine serum albumin

BSP

Bone sialoprotein

o

Degrees Celsius

C

Ca2+

Calcium ion

[Ca2+]i

Inorganic calcium

CAD

C-terminal TAD

CC

Calcified cartilage

CCDC80

Coiled-coil domain containing 80

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 

xiii 


 

CD9

Motility-related protein-1 (MRP-1)

CDS

Coding sequence

cDNA

Complementary dioxyribose nucleic acid

CGI

CpG island

ChIP

Chromatin immunoprecipitation

CHO

Chinese hamster ovary (cells)

C12orf29

Chromosome 12 open reading frame 29

CIAP

Calf intestinal alkaline phosphatase

CITED2

cAMP-responsive element-binding-protein-binding-protein CBP/p300
interacting-transactivators with glutamic acid and aspartic acid rich
tail

CNS

Central nervous system

COL1

Type I collagen

COL2

Type II collagen

CST3

Cystatin C

CSL

C-promoter binding factor 1 (CBF-1), suppressor of hairless (Su(H)),
lin-12 and glp-1 (Lag-1)

CTR

Calcitonin receptor

CyA

Cyclosporine A

dATP

Deoxyadenosine triphosphate

dCTP

Deoxycytidine triphosphate

ddH2O

Double distilled dihydrogen monoxide

DMP1

Dentin matrix protein-1

DEPC

Diethylpyrocarbonate

DSPP

Dentin sialophosphoprotein

Dex

Dexamethasone

dGTP

Deoxyguanosine triphosphate

DKK1

Dickkopf 1

DLX

Distalless homeobox protein

DMEM

Dulbecco’s Modified Eagle Medium

dNTP

Deoxynucleotide triphosphates

DNA

National Dyslexia Association

dpc

Days post coitum

Dpp

Decapentaplegia

xiv 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 


 

ds-cDNA

Double stranded cDNA

dTTP

Deoxythymidine triphosphate

DV

Dorsoventral

E-box

Enhancer box

E.coli

Escherichia coli

ECL

Enhanced chemiluminescence

ECM

Extracellular matrix

EDTA

Ethylenediaminetetraacetic acid

EMSA

Electrophoretic mobility shift assay

EMT

Epithelial to mesenchymal transition

En-1

Engrailed 1

ER

Endoplasmic reticulum

ERAD

ER-associated protein degradation

EST

Expressed sequence tag

EtBr

Ethidium bromide

EtNP

Ethanolamine phosphate

EtOH

Ethanol

FCS

Foetal calf serum

FGFs

Fibroblast growth factors

Fwd

Forward

g

Gram

G

Guanine

Ga

Giga annum

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

GDP

Guanosine diphosphate

GOE

Great oxygenation event

GOI

Gene of interest

GPI

Glycosylphosphatidyl inositol

GRB

Genome regulatory block

GS domain

Glycine-serine domain

GTP

Guanosine triphosphate

h

hour(s)

HA tag

Hemagglutinin tag

HBAR

Heat-based antigen retrieval

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 

xv 


 

HCl

Hydrogen chloride

HCNE

Highly conserved noncoding elements

HERPUD1

Homocysteine-inducible, endoplasmic reticulum stress-inducible,
ubiquitin-like domain member 1

HES-1

Hairy and enhancer of split 1

HF

High fidelity

HH

Hamburger-Hamilton

HIF- 

Hypoxia inducing factor-alpha

HMG

High-mobility group

HOX

Homeobox

HP

Hydrostatic pressure

HRF

Histamine releasing factor

HRP

Horseradish peroxidise

H2O2

Hydrogen peroxide

HSP

Heat shock protein

HSPs

Hereditary spastic paraplegias

HUVEC

Human umbilical vein endothelial cells

iAs

Inorganic arsenic

IF

Immunofluoresence

IgE

Immunoglobulin E

IGF

Insulin-like growth factor

IHH

Indian hedgehog

IHP

Intermittent hydrostatic pressure

IL2

Interleukin-2

ISGC

International Sheep Genome Consortium

IVD

Intervertebral discs

IVSAs

In vitro splicing assays

Kan

Kanamycin

kb

Kilobases

kDa

Kilodalton

KFS

Klippel-Feil syndrome

KLF

Kruppel-like factor

LB

Luria broth

LB-amp

Ampicillin Luria broth

xvi 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 


 

LEF

Lymphoid enhancer factor

LRP

Low-density-lipoprotein receptor related protein

Ma

Mega annum

MACH

Method for Amplification and isolation of Complementaty DNA from
plasmid libraries that require no Hybridization

MALDI-TOF Matrix-assisted laser desorption/ionization time-of-flight
Man

Mannose

max

Maximum

MAX

Myc-associated factor X

MCMC

Markov chain Monte Carlo

MCS

Multiple cloning site

MFH1

Mesenchyme forkhead-1

MNCs

Mononuclear cells

ME

Maxillary expansion

MERF

Medical Engineering Research Facility

Met

Methionine

min

minutes

mg

Milligram

mL

Milliliter

ML

Maximum Likelihood

mm

Millimetres

mM

Millimolar

MMLV

Moloney murine leukemia virus

MMPs

Matrix metalloproteinases

mOBs

Mandible osteoblasts

mRNA

Messenger ribose nucleic acid

MSX

Muscle segmentation homeobox

mTOR

Mammalian target of rapamycin

MTs

Microtubules

MT1-MMP

Membrane type 1 matrix metalloproteinase

MW

Molecular weight

Myr

Million years

g

Microgram

L

Microliter

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 

xvii 


 

MVs

Matrix vesicles

N

Normal (acid/base)

NaAc

Sodium acetate

NaCl

Sodium chloride

NAD

N-terminal TAD

NaOH

Sodium hydroxide

NCBI

National Center for Biotechnology Information

NCC

Non-calcified cartilage

NEB

New England Biolabs

NF-kB

Nuclear factor-kappa beta

ng

Nanogram

NICD

Notch intracellular domain

NIH

National Institute of Health

NJ

Neighbor-Joining

NKX3.2

NK3 homeobox 2

nM

Nanomolar

NMD

Nonsense-mediated mRNA decay

NOE

Neoproterozoic oxygenation event

NP

Nucleus pulposus

NSW

New South Wales

nt

Nucleotide

OA

Osteoarthritis

OMIM

Online Mendelian Inheritance in Man

ON

Overnight

OP1

Osteogenic protein 1

OTX

Osterix

PAGE

Polyacrylamide gel electrophoresis

PAL

Present atmospheric level

PAS

Polyadenylation sites

PAX1

Paired box protein 1

PBMC

Peripheral blood mononuclear cells

PBS

Phosphate buffered saline

Pbx1

Pre-B-cell leukemia transcription factor 1

PCR

Polymerase chain reaction

xviii 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 


 

PD

Proximodistal

PFA

Paraformaldehyde

pg

Picogram

PHA

Phytohemaglutinin

PIGF

Phosphatidylinositol glycan anchor biosynthesis, class F

Pitx1

Paired-like homeodomain 1

PNK

Polynucleotide kinase

Pol

Polymerase

poly(A)+

Polyadenylated (mRNA)

PPi

Inorganic pyrophosphate

PPIA

Peptidylprolyl isomerase A

PRDX5

Peroxiredoxin 5

PS

Primitive streak

PSM

Presomitic mesoderm

PTHrP

Parathyroid hormone-related peptide

RA

Retinoic acid

rATP

Ribose adenosine triphosphate

RE

Restriction enzyme

Rev

Reverse

RNAse H

Ribonuclease H (hybrid)

RNPS1

Binding protein S1, serine rich domain

rpm

Revolutions per minute

RRM

RNA recognition motif

ROS

Reactive oxygen species

RNA

Ribonucleic acid

RNAi

RNA interference

RT

Room temperature

RT-qPCR

Real time quantitative PCR

RUNX2

Runt related gene 2

s

Seconds

SCB

Subchondral bone

SDS

Sodium dodecyl sulfate

SFRP

Secreted frizzled-related protein

SHH

Sonic hedgehog

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 

xix 


 

SIBLING

small integrin-binding ligand N-linked Glycoproteins

SLIP

Self-Ligation of Inverse PCR Products

SN

Supernatant

snRNPs

Small nuclear ribonucleoprotein particles

snSNP

Nonsynonymous single nucleotide polymorphism

SOG

Short order gastrulation

SOX

Sry-type high mobility group box

SP1

Specificity protein 1

SPG21

Spastic paraplegia 21

SR

Serine/argenine-rich

SRY

Sex-determining region Y

SSP1

Secreted phosphoprotein 1

STE buffer

Salt-Tris-EDTA buffer

T

Thymidine

TBP

TATA box binding protein

TBST

Tris buffered saline-Tween

TCF

T cell-specific transcription factor

TD-PCR

Touchdown PCR

TF

Transcriptional factor

TGF-

Transforming growth factor beta

Tm

Melting temperature

tOBs

Tibial osteoblasts

TPT1

Tumor protein, translationally controlled 1

TSC

Tuberous sclerosis

TSS

Transcription start site

un

Undulated

un-ex

Undulated-extensive

un-s

Undulated short-tail

UPF

Eukaryotic up-frameshift protein

UPR

Unfolded protein response

UTR

Untranslated region

UV

Ultraviolet

VIC

Victoria

VEGF

Vascular endothelial growth factor

xx 

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 


 

VIM

Vimentin

vol

Volume

vnd

Ventral nervous system defective

v/v

Volume per volume

WAT

White adipose tissue

WB

Western blot

Wg

Wingless (Drosophila)

WMIHC

Whole mount immunohistochemistry

WMISH

Whole mount in situ hybridization

WNT

Wingless

w/v

weight per volume

xg

times gravity

ZPA

Zone of polarizing activity

The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan 

xxi