Secondary Palate Development in the Dog (Canis lupus familiaris).

OBJECTIVE: To investigate the gestational timing of morphologic events in normal canine secondary palate development as a baseline for studies in dog models of isolated cleft palate (CP). METHODS: Beagle and beagle/cocker spaniel-hybrid fetal dogs were obtained by cesarean-section on various days of gestation, timed from the initial rise of serum progesterone concentration. Morphology of fetal heads was determined by examining serial coronal sections. RESULTS: On gestational day 35 (d35), the palatal shelves pointed ventrally alongside the tongue. On d36, palatal shelves were elongated and elevated to a horizontal position above the tongue but were not touching. On d37, palatine shelves and vomer were touching, but the medial epithelial seam (MES) between the apposed shelves remained. Immunostaining with epithelial protein markers showed that the MES gradually dissolved and was replaced by mesenchyme during d37-d44, and palate fusion was complete by d44. Examination of remnant MES suggested that fusion of palatal shelves began in mid-palate and moved rostrally and caudally. CONCLUSION: Palate development occurs in dogs in the steps described in humans and mice, but palate closure occurs at an intermediate time in gestation. These normative data will form the basis of future studies to determine pathophysiologic mechanisms in dog models of CP. Added clinical significance is the enhancement of dogs as a large animal model to test new approaches for palate repair, with the obvious advantage of achieving full maturity within 2 years rather than 2 decades.

Imputation of canine genotype array data using 365 whole-genome sequences improves power of genome-wide association studies.

Genomic resources for the domestic dog have improved with the widespread adoption of a 173k SNP array platform and updated reference genome. SNP arrays of this density are sufficient for detecting genetic associations within breeds but are underpowered for finding associations across multiple breeds or in mixed-breed dogs, where linkage disequilibrium rapidly decays between markers, even though such studies would hold particular promise for mapping complex diseases and traits. Here we introduce an imputation reference panel, consisting of 365 diverse, whole-genome sequenced dogs and wolves, which increases the number of markers that can be queried in genome-wide association studies approximately 130-fold. Using previously genotyped dogs, we show the utility of this reference panel in identifying potentially novel associations, including a locus on CFA20 significantly associated with cranial cruciate ligament disease, and fine-mapping for canine body size and blood phenotypes, even when causal loci are not in strong linkage disequilibrium with any single array marker. This reference panel resource will improve future genome-wide association studies for canine complex diseases and other phenotypes.

Persistent Mullerian duct Syndrome in a Brazilian miniature schnauzer dog.

Here we describe an eight-year-old miniature schnauzer (MS) dog from Brazil with Persistent Mullerian Duct Syndrome (PMDS) and the single base pair substitution in AMHR2 exon 3, first detected in this breed in the USA. This finding is evidence of mutation dissemination to South America. In PMDS, a type of XY Disorder of Sex Development (DSD), dogs with a male karyotype and external phenotype also have a uterus, oviducts, and a cranial vagina internally. Approximately half of PMDS MS are unilaterally or bilaterally cryptorchid and many develop pyometra and/or Sertoli cell tumor. Bilateral Sertoli cell testicular tumors were present in this case, and the dog died a few days after surgical castration and hysterectomy. Although the karyotype was compatible with male chromosomal sex, a Robertsonian translocation was also identified, which may be an incidental finding. This report emphasizes the importance of cytogenetic and molecular genetic analyses, along with clinical examination, to identify chromosomal or genetic variations associated with canine PMDS. These are helpful tools to obtain early diagnosis in the MS, which is important to improve health outcomes for affected dogs and to reduce the prevalence of PMDS and cryptorchidism in this breed by avoiding the mating of carriers.

BarkBase: Epigenomic Annotation of Canine Genomes.

Dogs are an unparalleled natural model for investigating the genetics of health and disease, particularly for complex diseases like cancer. Comprehensive genomic annotation of regulatory elements active in healthy canine tissues is crucial both for identifying candidate causal variants and for designing functional studies needed to translate genetic associations into disease insight. Currently, canine geneticists rely primarily on annotations of the human or mouse genome that have been remapped to dog, an approach that misses dog-specific features. Here, we describe BarkBase, a canine epigenomic resource available at barkbase.org. BarkBase hosts data for 27 adult tissue types, with biological replicates, and for one sample of up to five tissues sampled at each of four carefully staged embryonic time points. RNA sequencing is complemented with whole genome sequencing and with assay for transposase-accessible chromatin using sequencing (ATAC-seq), which identifies open chromatin regions. By including replicates, we can more confidently discern tissue-specific transcripts and assess differential gene expression between tissues and timepoints. By offering data in easy-to-use file formats, through a visual browser modeled on similar genomic resources for human, BarkBase introduces a powerful new resource to support comparative studies in dogs and humans.

Gene expression in hip soft tissues in incipient canine hip dysplasia and osteoarthritis.

Canine hip dysplasia and developmental dysplasia of the human hip share demographic, phenotypic, and clinical features including the predisposition to develop osteoarthritis in affected joints. To support the results of genetic mapping studies for CHD and its concomitant osteoarthritis with functional information, we performed RNA-seq on hip capsule and teres ligament of affected and unaffected dogs. RNA seq showed that expressed genes segregated according age, capsule or ligament, and hip phenotype. Expression of HHIP, DACT2, and WIF1 was significantly higher in capsule from control hips than dysplastic hips indicating a disruption of the hedgehog signaling pathway. Expression of SPON 1, a key component of the WNT pathway, was increased significantly in both dysplastic capsule and ligament while FBN2 and EMILIN3 were significantly increased in dysplastic capsule. Of genes associated with human hip osteoarthritis, expression of ACAN, IGF1, CILP2, COL11A1, COL8A1, and HAPLN was increased significantly in dysplastic capsule. The significant increase in expression of PLA2F, TNFRSF, TMEM, and IGFBP in dysplastic capsule indicated an injury response. Gene set enrichment analysis revealed that genes involved in extracellular matrix structure, epithelial to mesenchymal transition, myogenesis, growth factor signaling, cancer and immune pathways were enriched in dysplastic capsule. For teres ligament from dysplastic joints, genes in retinoic signaling pathways and those encoding extracellular matrix molecules, but not proteoglycans, were enriched. Hip tissues respond to abnormal mechanics early in dysplastic hip development and these pathways present targets for intervention in the early synovitis and capsulitis secondary to canine and human hip dysplasia. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:313-324, 2019.

XX Disorder of Sex Development is associated with an insertion on chromosome 9 and downregulation of RSPO1 in dogs (Canis lupus familiaris).

Remarkable progress has been achieved in understanding the mechanisms controlling sex determination, yet the cause for many Disorders of Sex Development (DSD) remains unknown. Of particular interest is a rare XX DSD subtype in which individuals are negative for SRY, the testis determining factor on the Y chromosome, yet develop testes or ovotestes, and both of these phenotypes occur in the same family. This is a naturally occurring disorder in humans (Homo sapiens) and dogs (C. familiaris). Phenotypes in the canine XX DSD model are strikingly similar to those of the human XX DSD subtype. The purposes of this study were to identify 1) a variant associated with XX DSD in the canine model and 2) gene expression alterations in canine embryonic gonads that could be informative to causation. Using a genome wide association study (GWAS) and whole genome sequencing (WGS), we identified a variant on C. familiaris autosome 9 (CFA9) that is associated with XX DSD in the canine model and in affected purebred dogs. This is the first marker identified for inherited canine XX DSD. It lies upstream of SOX9 within the canine ortholog for the human disorder, which resides on 17q24. Inheritance of this variant indicates that XX DSD is a complex trait in which breed genetic background affects penetrance. Furthermore, the homozygous variant genotype is associated with embryonic lethality in at least one breed. Our analysis of gene expression studies (RNA-seq and PRO-seq) in embryonic gonads at risk of XX DSD from the canine model identified significant RSPO1 downregulation in comparison to XX controls, without significant upregulation of SOX9 or other known testis pathway genes. Based on these data, a novel mechanism is proposed in which molecular lesions acting upstream of RSPO1 induce epigenomic gonadal mosaicism.

Live Births from Domestic Dog (Canis familiaris) Embryos Produced by In Vitro Fertilization.

Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology. This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies-an approach that would also advance the dog as a biomedical model. Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species. Here we report the first live births from in vitro fertilized embryos in the dog. Adding to the practical significance, these embryos had also been cryopreserved. Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis. We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg. Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4-5 after the luteinizing hormone (LH) surge. We found that only on Day 6 are oocytes consistently able to be fertilized. In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146). Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies. Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species.

A single base pair mutation encoding a premature stop codon in the MIS type II receptor is responsible for canine persistent Müllerian duct syndrome.

Müllerian inhibiting substance (MIS), a secreted glycoprotein in the transforming growth factor-beta family of growth factors, mediates regression of the Müllerian ducts during embryonic sex differentiation in males. In persistent Müllerian duct syndrome (PMDS), rather than undergoing involution, the Müllerian ducts persist in males, giving rise to the uterus, fallopian tubes, and upper vagina. Genetic defects in MIS or its receptor (MISRII) have been identified in patients with PMDS. The phenotype in the canine model of PMDS derived from the miniature schnauzer breed is strikingly similar to that of human patients. In this model, PMDS is inherited as a sex-limited autosomal recessive trait. Previous studies indicated that a defect in the MIS receptor or its downstream signaling pathway was likely to be causative of the canine syndrome. In this study, the canine PMDS phenotype and clinical sequelae are described in detail. Affected and unaffected members of this pedigree are genotyped, identifying a single base pair substitution in MISRII that introduces a stop codon in exon 3. The homozygous mutation terminates translation at 80 amino acids, eliminating much of the extracellular domain and the entire transmembrane and intracellular signaling domains. Findings in this model could enable insights to be garnered from correlation of detailed clinical descriptions with molecular defects, which are not otherwise possible in the human syndrome.

Parturition prediction and timing of canine pregnancy.

An accurate method of predicting the date of parturition in the bitch is clinically useful to minimize or prevent reproductive losses by timely intervention. Similarly, an accurate method of timing canine ovulation and gestation is critical for development of assisted reproductive technologies, e.g. estrous synchronization and embryo transfer. This review discusses present methods for accurately timing canine gestational age and outlines their use in clinical management of high-risk pregnancies and embryo transfer research.

Genetics, genomics, and molecular biology of sex determination in small animals.

The genomic revolution is beginning to facilitate advances in canine and feline medicine, as illustrated in our research. Our studies are focused upon identifying the gene mutation that causes canine Sry-negative XX sex reversal, a disorder of sex determination in which chromosomal females (78,XX) develop testicular tissue, becoming either XX true hermaphrodites with ovotestes, or XX males with bilateral testes. A genome-wide screen, using mapped markers in our pedigree of Sry-negative XX sex reversed dogs founded upon the American cocker spaniel, identified five chromosomal regions in which the causative gene may be located. The canine genome was used to identify the canine homologue of goat Pisrt1 and so determine that canine and caprine Sry-negative XX sex reversal are genetically heterogeneous. A second goal of our research is to determine the molecular mechanism by which the mutation causes testis induction. Thus far, we have reported gonadal Sry and Sox9 expression patterns in normal embryos, which have temporal and spatial patterns similar to those reported in humans, sheep, and pigs. Once gene mutations causing such inherited disorders are identified, DNA tests will become a part of general veterinary practice, advancing both diagnostic techniques and preventative medicine.

Sf1 and Mis expression: molecular milestones in the canine sex determination pathway.

In mammals, the Y-linked Sry gene is normally responsible for testis induction. However, testes develop in the absence of Sry in human patients and animal models with Sry-negative XX sex reversal. The mechanism of testis induction in this disorder is presently unknown. Characterization of gene expression in normal embryos contributes to the framework within which the canine Sry-negative XX sex reversal model can be evaluated. The objective of this study was to add two molecular milestones to the canine sex determination pathway by determining the temporal and spatial expression patterns of Sf1 and Mis in normal urogenital ridges (UGR) at various gestational stages. The onset of Sf1 expression signifies the start of the sex determination period, whereas initial Mis expression identifies the end of the testis induction period. Sf1 expression in UGR was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and whole mount in situ hybridization (WMISH) at Carnegie stages (CS) 15 to 20. Canine sex determination begins at CS 15 with Sf1 expression in the emerging indifferent gonad. Gonadal Sf1 expression was detected in both sexes at all ages, and in the presumptive adrenal primordium at CS 15 and 17. At stages > or = CS 17, Sf1 expression was pronounced in male and female gonads. Mis expression, assayed by WMISH at CS 13.5-20, was observed only in male gonads > or = CS 18, indicating that the testis induction period ends at CS 18. The expression patterns of both genes are similar to those observed in humans and domestic animals.

Accuracy of canine parturition date prediction using fetal measurements obtained by ultrasonography.

The length of canine gestation is 65 days from the luteinizing hormone (LH) surge. Early and accurate determination of canine gestational age is useful for predicting and managing parturition. We performed a retrospective study on fetal measurements obtained by transabdominal ultrasonographic examination of 83 bitches (32 breeds) to estimate gestational age. Gestational age was estimated using two published tables correlating either (1). embryonic vesicle diameter (EVD), crown-rump length (CRL), body diameter (BD), and biparietal diameter (HD) to the LH surge in mid-gestational beagles or (2). BD and HD to parturition in late-gestation retrievers. Parturition date was predicted by obtaining the difference between the gestational age estimate and 65 days. Bitches were divided into four body weight (BW) groups based on nonpregnant body weight: small (9-20 kg), large (>20-40 kg), and giant (>40 kg). Mean+/-S.D. litter size (LS) was calculated for each BW group. The BW groups were then divided into small, average, or large LS groups. The accuracy of the prediction was not affected by LS but was affected by maternal body weight for small and giant BW groups only. When adjusted for weight, the accuracy of prediction within +/-1 day and +/-2 day intervals was 75 and 87%, respectively. Using stepwise logisitic regression, the most accurate prediction of parturition date was obtained when fetuses were measured at 30 days after the LH surge, regardless of body weight or LS. Parturition date predictions made after 39 days of gestation using only biparietal and BD fetal measurements were <50% accurate within +/-2 days.

Accuracy of canine parturition date prediction from the initial rise in preovulatory progesterone concentration.

Accurate prediction of parturition date is useful for clinical management of canine parturition. For nearly all normal canine pregnancies, parturition occurs 64-66 days from the LH peak, the timing of which cannot be differentiated from the initial sharp rise in serum progesterone (P4) concentrations. We sought to determine by retrospective analysis if prebreeding serum progesterone concentrations could accurately predict parturition date. Serum progesterone concentrations recorded as serial samples from 63 bitches (19 breeds) were analyzed. Progesterone concentrations were measured by radioimmunoassay (RIA) or chemiluminescent immunoassay (CLIA). The CLIA method was validated for use in determining P4 concentrations in canine serum and results were comparable to those obtained with RIA. Bitches were grouped by nonpregnant body weight (BW) and litter size (LS). Day 0 (D0), the day of preovulatory rise in serum P4, was defined as the day that P4 concentration rose to > or =l.5 ng/ml and was at least twice the baseline concentration. The predicted parturition date, 65 days following the day of preovulatory rise in serum P4 (D65), was compared to actual parturition date, the day the first pup was delivered. We determined that mean P4 concentration at D0 for all BW groups was 2.02+/-0.18 ng/ml and there was significant variation in P4 concentrations between BW groups after D1. In addition, we determined that the accuracy of parturition date prediction within a +/-1, +/-2, and +/-3 day interval using prebreeding serum progesterone concentrations was 67, 90, and 100%, respectively, and that the accuracy was not affected by body weight or litter size.