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This Wiki page is a summation of work done by Schoenebeck, Jeffery J, et al which uses various techniques to study the morphology of canine skulls.

Dog-breeds.jpg

Background

Domestication of dogs is thought to have begun between 11 and 16 thousand years ago when ancestors of modern dogs started living in closer proximity to humans. The exact domestication process is unknown. In 2013 the World Canine Organization recognized 339 breeds of dogs, but this is believed to be an underestimation (Coren et. al. 2013). It is quite remarkable that such differentiation within the dog species has been observed since the idea of different breed standards, and breed clubs, only happened in the 1800s. The dramatic increase in types of breeds happened quite recently. Scientists can even distinguish between breeds because each one has a unique DNA signature. The many classes of dog breeds have come to be with the help of human artificial selection. Humans have created extremely small, and extremely large dogs; dogs with significant health problems, and dogs with anatomically problematic skeletal structures. A prominent example is the diversification of skull structure types. Breeds like the pug and bulldog have brachycephalic skulls, and breeds like the greyhound have dolichocephalic skulls (Pickrell 2004).

What is brachycephaly and dolichocephaly?

Figure 1

Canines have diverged into distinguished breeds based largely on skull classifications. Over time breeders have selected for specific, advantageous traits in dogs, causing the isolation of interbred dogs to form distinct skull shapes. Of the three major skull structures, this research paper focuses on the two extremes: brachycephalic and dolichocephalic. Skull classifications are done by taking two measurements, one of the cranium and one of the nasal cavity. Dolichocephalic skulls have an elongated nasal cavity which extends from a shorter cranium (Figure 1). The extended snout of dolichocephalic breeds enhances their sense of smell and makes them superior hunting dogs. Brachycephalic skulls are notoriously flat with the cranium having a much larger measurement compared to the short or even nonexistent nasal cavity (Figure 2) (Writer, 2010). There are no known advantages for breeds with the brachycephalic skulls, but there are numerous health hazards. For example, figure 3 demonstrates the receding development of the nasal cavity for the British Bulldog over the past fifty years. Despite the reduction in space, the amount of soft tissue in the nasal cavity remains constant. The overcrowded compaction of the soft tissue causes severe breathing problems for dogs with undersized snouts (McAlinden, 2012).

Figure 2
Figure 3


Dolichocephaly is considered the wild type phenotype, and is also the most similar to the common ancestor the wolf. Brachycephaly, on the other hand, is a mutant phenotype caused by stunted growth factors in the developing skull. The genetic factors identified to be responsible for differentiating skull development were found by conducting Genome Wide Association Studies (GWAS) and by using Single Nucleotide Polymorphism (SNP) chips to carry out mapping studies on the dog genome. Mutations in the muscle encoded transcription factors: TWIST1 and MSX2, along with inhibited binding for fibroblast growth factors (FGFs) are notable genetic defects that result in the development of brachycephalic skulls. In addition to FGFs, there is another paracrine factor: TGF-B induces changes in neighboring cells to develop and maintain sutures and synchondroses, which are critical for skull growth. Sutures are tight, immovable junctions between bones, while synchondroses are cartilage that either forms joints between bones or forms bones during development. Coronal synostosis occurs during infancy when fibrous structures fuse prematurely to form bone in the skull. Although coronal synostosis is usually absent in dogs, the cranial bases of both brachycephalic and dolichocephalic breeds are disproportional to the rest of their bodies. This indicates that synchondroses play an important role in skull growth since their fusion and development affect the formation of the skull. The bone morphogenetic protein 3 (BMP3) has also been found to affect dolichocephalic and brachycephalic skull formation by inhibiting TGF-B and being highly expressed in synchondroses. Tying in all the components found to affect skull development in dogs, BMP3 prevents osteogenesis, the brittle bone disease, by its presence in synchondroses. In synchondroses, BMP3 assists with the development of cartilage which will form into bone and shape the skull (Schoenebeck and Ostrander, 2013).

What is the BMP-3 Gene?

Bone Morphogenetic Proteins (BMPs) consist of several different proteins that have a common function: they are growth factors. They belong to the transforming growth factor β superfamily (shortened to the acronym TGFβ) (Chen, 2004). BMPs differ from other TGF-B members by having two less conserved cysteines than the TGF-B members. They were given the name Bone Morphogenic Proteins because at the time they were discovered, they were exhibiting their ability to form bones. In reality, under different conditions, they have a wide variety of functions (Gilbert, 2010). In addition to bone formation, they also influence cardiac, neural, and cartilage development (Chen, 2004). BMP-3 is one of the many proteins in this classification, and its specific function involves the development, growth, and healing of bone. It is the most abundant protein to be found in the bone matrix, making up 65% of all BMPs found (Mcmahon, 2012). Information regarding this gene seems contradictory and its fully spectrum of functions are not entirely known or understood. One study involving mice showed this gene to be a negative regulator of bone density. Mice were generated to lack the BMP3 gene had double the amount of trabecular bone than their littermates that did contain the BMP3 gene (Daluski, 2001). Another study on zebrafish seems to have shown the opposite. Focusing now on the influences BMP3 has on craniofacial development, BMP3 was knocked down in zebrafish and the resulting phenotype was a severe lack of jaw formation due to the lack of production of cartilage. One explanation for this contradiction of information can be attributed to the fact that the study on the mice did not report the effects on the cranial bones. Regardless, two things can reasonably be determined from this information: the BMP3 gene has roles in bone development (and to our interest, roles in craniofacial development) and is a conserved gene that is found in many organisms. An ortholog of this gene has been examined in canines and has been determined to account for one of the determinants for the differences in skulls of dog breeds; a missense mutation in the BMP3 gene of dogs has lead to the brachycephalic phenotype. (Schoenebeck, 2012).

Summary of the Study by Schoenebeck

In the study “Variation of BMP3 Contributes to Dog Breed Skull Diversity” researchers tried to determine the gene(s) that have a hand in the understanding of control of cranial shape variations, which is poorly understood at this time. They studied canine skulls using a Genome Wide Association Study (GWAS) that combined the genetic profiles of 576 purebred dogs (representing 62 breeds) with craniometric breed-sex averages collected from skulls from 533 canines (representing 120 dog breeds and four subspecies of gray wolf) and were together assayed using SNP chips. From this scan, the researchers identified five quantified trait loci (QTL) that were likely candidates for the cranioskeletal differences between dolichocephalic and brachycephalic breeds of dogs. The most notable of these was at the CFA32 QTL, a missense mutation in BMP3 that occurs in small brachycephalic breeds (Schoenebeck, 2012).

GWAS

Genome Wide Association Studies are a method of gene identification. Entire genomes of many subjects are scanned to find genetic variations that are associated with a particular trait or disease. This is carried out by placing each purified genome on chips and scanning with machines that detect single nucleotide polymorphisms (SNPs), which are markers for genetic variation. Significant variations indicate associated with a certain trait which can then be further tested through sequencing or other assays ("Genome-Wide Association Studies Fact Sheet," 2014). In the study on canine cranioskeletal development, researchers scanned for PC1 association. PC1 is a component of shape variance that defines the changes between dolichocephalic and brachycephalic breeds. Once false associations from breed relatedness, as well as associations based on whole-body size, were removed, 5 significant and stable QTL were found to account for skull shape changes in relation to PC1. These five QTLs were on CFA1, 5, 24, 32, and X (Schoenebeck, 2012).

What They Found

Of the five QTL's identified, researchers decided to focus on CFA32 because it showed strong evidence of selection, and this region had never been studied before. This region contains only two genes, cGMP-dependent protein kinase 2 (PRKG2) and bone morphogenetic protein 3 (BMP3), that were examined as potential regulators of brachycephalic skull development. Whole-genome sequence analysis was used on 11 different dog breeds with varying skull shapes, including Pekingese and Bulldog since they are in the brachycephaly skull group. An SNP was discovered among the genomes that encoded a missense mutation in the BMP3 gene. The mutation changed a phenylalanine to a leucine at position 452 (BMP3F453L). Position 452 is usually occupied by an aromatic amino acid, so changing the structure to a leucine seems to restrict bone growth. The specific mechanism is undetermined but it is thought that BMP3 inhibits other BMPs and Activins by binding to the ActRIlb receptor. This mutation appeared to be fixed among brachycephalic breeds when looking at the genotypes of 842 dogs from 113 breeds (Schoenebeck, 2012).

BMP3 In Zebrafish

Researchers wanted to confirm BMP3s role in skull development so they assayed its function in zebrafish. Zebrafish is a model that develops quickly and it’s easy to knock down their gene expression. The BMP3 homolog for zebrafish was identified on chromosome 5. To test bmp3 expression, researchers knocked down BMP3 activity by injecting translation-blocking antisense morpholino oligonucleotides (MO). Embryos injected with MO's had severe jaw development deficiencies and loss of cartilage. These results indicate BMP3's role in craniofacial development and that this is an ancient process (Schoenebeck, 2012).

Implications

In a study involving the BMP2 gene and the BMP3 researchers discovered the antagonistic behavior of BMP3 to BMP2 in regards to its involvement with cranial bone growth. They discovered that BMP3 competes with BMP2 for the binding site of a common effector, SMAD4. BMP3 will suppress osteoblastic differentiation and limit differentiation of osteoprogenitors, such as suture cells. Knowing that BMP2 and BMP3 are both expressed in the cranial cells and that the suture mesenchymal cells respond highly to BMP2 and BMP3, scientists may be able to change the outcome of abnormal cranial bone growth in conditions like craniosynostosis (Dwivedi et. al. 2012). Craniosynostosis is a birth defect that results from a premature fusion of the joints between bones of a baby’s skull, and it results in the brain not being able to grow to its natural shape; the head is very misshapen (Mayo Clinic Staff).

The BMP3 gene, along with three other genes, has also been associated with colorectal cancer. They believe it is the inactivation of the BMP3 gene that leads to colorectal tumor development. When screening for colorectal neoplasia the BMP3 gene is methylated, and thus silenced. The BMP3 gene has also been found to be a good methylation marker in stool assays for detection of pancreatic cancer (Wang et. al. 2014).

References

  1. Coren, Stanley. "How Many Breeds of Dogs Are There in the World?"Psychology Today. N.p., 23 May 2013. Web. 01 Mar. 2015.
  2. Pickrell, John. "Dog DNA Study Yields Clues to Origins of Breeds." National Geographic. National Geographic Society, 20 May 2004. Web. 01 Mar. 2015.
  3. Writer, Staff. "Dog Skull Shapes." MetroSniff. 9 Aug. 2010. Web. 01 Mar. 2015. <http://www.metrosniff.com/content/dog-skull-shapes>.
  4. McAlinden, Aidan. "Brachycephalic Dog Breeds." The Veterinary Expert Pet Health. 25 Nov. 2012. Web. 01 Mar. 2015. <http://www.theveterinaryexpert.com/nose-and-throat/what-is-a-brachycephalic-dog/>.
  5. Schoenebeck, Jeffrey J., and Elaine A. Ostrander. "The Genetics of Canine Skull Shape Variation." Genetics. Genetics Society of America, Feb. 2013. Web. 01 Mar. 2015. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567726/>.
  6. Chen, Di, et. al. "Growth Factors." Bone Morphogenetic Proteins, Informa Healthcare. 2004. Web. 01 Mar. 2015. <http://informahealthcare.com/doi/abs/10.1080/08977190412331279890>.
  7. Gilbert, Scott F. (2010). Developmental Biology (9th ed.). Sunderland, MA: Sinauer Associates, Inc.
  8. McMahon, Mark S. "Bone Morphogenic Protein 3 Signaling in the Regulation of Osteogenesis." Healio Orthopedics. Orthopedics, Nov. 2012. Web. 01 Mar. 2015. <http://www.healio.com/orthopedics/journals/ortho/2012-11-35-11/%7B35780911-3f64-4d41-bc38-809645b340b6%7D/bone-morphogenic-protein-3-signaling-in-the-regulation-of-osteogenesis>.
  9. Daluski, Aaron. "Bone Morphogenetic Protein-3 Is a Negative Regulator of Bone Density." Nature. Nature Genetics, 2001. Web. 1 Mar. 2015. <http%3A%2F%2Fwww.nature.com%2Fng%2Fjournal%2Fv27%2Fn1%2Ffull%2Fng0101_84.html>.
  10. Schoenebeck, Jeffery J, et. al. "Variation of BMP3 Contributes to Dog Breed Skull Diversity." PLOS Genetics, 2 Aug. 2012. Web. 01 Mar. 2015. <http://journals.plos.org/plosgenetics/article?id=10.1371%2Fjournal.pgen.1002849>.
  11. "Genome-Wide Association Studies Fact Sheet." NIH. National Human Genome Research Institute, 27 Jan. 2014. Web. 25 Feb. 2015. <http://www.genome.gov/20019523>.
  12. Dwivedi, Prem P., Peter J. Anderson, and Barry C. Powell. "Development of an Efficient, Non-viral Transfection Method for Studying Gene Function and Bone Growth in Human Primary Cranial Suture Mesenchymal Cells Reveals That the Cells Respond to BMP2 and BMP3." BMC Biotechnology 12.1 (2012): 45. Web. 1 Mar. 2015.
  13. Mayo Clinic Staff. "Craniosynostosis." Definition. Mayo Clinic, n.d. Web. 01 Mar. 2015.
  14. Wang, Richard M., Jordan Green, and Zhongliang Wang. "Bone Morphogenetic Protein (BMP) Signaling in Development and Human Diseases." Genes and Diseases 1.1 (2014): 87-105. ScienceDirect. Web. 1 Mar. 2015.