Shaping and interpretation of Dpp morphogen gradient by endocytic trafficking

Dpp/BMP is a morphogen that controls patterning and growth in the Drosophila wing disc. Contrast with the extracellular and nuclear regulation, how Dpp morphogen gradient is shaped and interpreted by endocytic trafficking remains unclear. To address this, here we generate novel fluorescent protein tagged dpp alleles that allow to visualize both extracellular and intracellular Dpp distribution. Using these alleles, we found that, while blocking endocytosis expanded the extracellular Dpp gradient and impaired Dpp signaling, blocking early endosome expanded not only the extracellular Dpp gradient but also Dpp signaling range due to impaired downregulation of activated receptors. We show that blocking multivesicular body (MVB) formation, but not late endosome, expanded Dpp signaling and caused accumulation of the intracellular Dpp without affecting the extracellular Dpp gradient. These results indicate that, while the early endocytosis acts as a sink for Dpp and initiates Dpp signaling, termination of Dpp signaling at MVB is required for interpretation of the extracellular Dpp gradient. Taken together, our results reveal that extracellular Dpp morphogen gradient is shaped and interpreted by distinct endocytic trafficking pathways

Multiple FGF4 retrocopies recently derived within canids

Two transcribed retrocopies of the fibroblast growth factor 4 (FGF4) gene have previously been described in the domestic dog. An FGF4 retrocopy on chr18 is associated with disproportionate dwarfism, while an FGF4 retrocopy on chr12 is associated with both disproportionate dwarfism and intervertebral disc disease (IVDD). In this study, whole-genome sequencing data were queried to identify other FGF4 retrocopies that could be contributing to phenotypic diversity in canids. Additionally, dogs with surgically confirmed IVDD were assayed for novel FGF4 retrocopies. Five additional and distinct FGF4 retrocopies were identified in canids including a copy unique to red wolves (Canis rufus). The FGF4 retrocopies identified in domestic dogs were identical to domestic dog FGF4 haplotypes, which are distinct from modern wolf FGF4 haplotypes, indicating that these retrotransposition events likely occurred after domestication. The identification of multiple, full length FGF4 retrocopies with open reading frames in canids indicates that gene retrotransposition events occur much more frequently than previously thought and provide a mechanism for continued genetic and phenotypic diversity in canids

A second KRT71 allele in curly coated dogs.

Major characteristics of coat variation in dogs can be explained by variants in only a few genes. Until now, only one missense variant in the KRT71 gene, p.Arg151Trp, has been reported to cause curly hair in dogs. However, this variant does not explain the curly coat in all breeds as the mutant Trp allele, for example, is absent in Curly Coated Retrievers. We sequenced the genome of a Curly Coated Retriever at 22× coverage and searched for variants in the KRT71 gene. Only one protein-changing variant was present in a homozygous state in the Curly Coated Retriever and absent or present in a heterozygous state in 221 control dogs from different dog breeds. This variant, NM_001197029.1:c.1266_1273delinsACA, was an indel variant in exon 7 that caused a frameshift and an altered and probably extended C-terminus of the KRT71 protein NP_001183958.1:p.(Ser422ArgfsTer?). Using Sanger sequencing, we found that the variant was fixed in a cohort of 125 Curly Coated Retrievers and segregating in five of 14 additionally tested breeds with a curly or wavy coat. KRT71 variants cause curly hair in humans, mice, rats, cats and dogs. Specific KRT71 variants were further shown to cause alopecia. Based on this knowledge from other species and the predicted molecular consequence of the newly identified canine KRT71 variant, it is a compelling candidate causing a second curly hair allele in dogs. It might cause a slightly different coat phenotype than the previously published p.Arg151Trp variant and could potentially be associated with follicular dysplasia in dogs

Two MC1R loss-of-function alleles in cream-coloured Australian Cattle Dogs and white Huskies.

Loss-of-function variants in the MC1R gene cause recessive red or yellow coat-colour phenotypes in many species. The canine MC1R:c.916C>T (p.Arg306Ter) variant is widespread and found in a homozygous state in many uniformly yellow- or red-coloured dogs. We investigated cream-coloured Australian Cattle Dogs whose coat colour could not be explained by this variant. A genome-wide association study with 10 cream and 123 red Australian Cattle Dogs confirmed that the cream locus indeed maps to MC1R. Whole-genome sequencing of cream dogs revealed a single nucleotide variant within the MITF binding site of the canine MC1R promoter. We propose to designate the mutant alleles at MC1R:c.916C>T as e1 and at the new promoter variant as e2. Both alleles segregate in the Australian Cattle Dog breed. When we considered both alleles in combination, we observed perfect association between the MC1R genotypes and the cream coat colour phenotype in a cohort of 10 cases and 324 control dogs. Analysis of the MC1R transcript levels in an e /e compound heterozygous dog confirmed that the transcript levels of the e2 allele were markedly reduced with respect to the e1 allele. We further report another MC1R loss-of-function allele in Alaskan and Siberian Huskies caused by a 2-bp deletion in the coding sequence, MC1R:c.816_817delCT. We propose to term this allele e3. Huskies that carry two copies of MC1R loss-of-function alleles have a white coat colour

An ADAMTS3 Missense Variant is Associated with Norwich Terrier Upper Airway Syndrome

In flat-faced dog breeds, air resistance caused by skull conformation is believed to be a major determinant of Brachycephalic Obstructive Airway Syndrome (BOAS). The clinical presentation of BOAS is heterogeneous, suggesting determinants independent of skull conformation contribute to airway disease. Norwich Terriers, a mesocephalic breed, are predisposed to Upper Airway Syndrome (UAS), a disease whose pathological features overlap with BOAS. Our health screening clinic examined and scored the airways of 401 Norwich terriers by laryngoscopy. Genome-wide association analyses of UAS-related pathologies revealed a genetic association on canine chromosome 13 (rs9043975, p = 7.79x10-16). Whole genome resequencing was used to identify causal variant(s) within a 414 kb critical interval. This approach highlighted an error in the CanFam3.1 dog assembly, which when resolved, led to the discovery of a c.2786G>A missense variant in exon 20 of the positional candidate gene, ADAM metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3). In addition to segregating with UAS amongst Norwich Terriers, the ADAMTS3 c.2786G>A risk allele frequency was enriched among the BOAS-susceptible French and (English) Bulldogs. Previous studies indicate that ADAMTS3 loss of function results in lymphoedema. Our results suggest a new paradigm in the understanding of canine upper airway disease aetiology: airway oedema caused by disruption of ADAMTS3 predisposes dogs to respiratory obstruction. These findings will enhance breeding practices and could refine the prognostics of surgical interventions that are often used to treat airway obstruction

Role of cellular trafficking on Dpp morphogen gradient formation and signaling

Dpp/BMP is a well-studied morphogen that controls patterning and growth in the Drosophila wing disc. However, how the Dpp morphogen gradient is established and is interpreted by endocytic trafficking remains largely unknown. By utilizing the endogenously tagged dpp alleles with the monomeric proteins mGreenLantern and mScarlet, I investigated the role of different trafficking factors in shaping the intra- and the extracellular Dpp gradient. Using these alleles, I showed that dynamin is a major regulator of the Dpp gradient and blocking dynamin-dependent endocytosis expanded the extracellular Dpp gradient and impaired Dpp signaling. I also found that blocking the early endosomal trafficking by knocking down Rab5 not only expanded the extracellular Dpp gradient, but also increased the range of Dpp signaling possibly due to an impaired termination of its receptor Tkv. I also demonstrated that blocking multivesicular body (MVB) formation, but not the endo-lysosomal fusion, expanded the internalized Dpp distribution and signaling range without affecting the extracellular Dpp gradient. By investigating the role of recycling endosomes, I also showed that while the slow recycling endosomes slightly affected the intracellular Dpp distribution, the fast recycling endosomes minimally affected the extracellular Dpp gradient and neither of these factors influenced the Dpp signaling activity. My findings indicated that the early endocytic factors act as a sink for the extracellular Dpp gradient and are required to activate Dpp signaling, while the late endocytic factors terminate Dpp signaling activity by sorting the activated receptors into the intraluminal vesicles (ILVs). Taken together, our results suggest that extracellular Dpp morphogen gradient is shaped and interpreted by distinct endocytic trafficking pathways

Multiple FGF4 Retrocopies Recently Derived within Canids.

Two transcribed retrocopies of the fibroblast growth factor 4 (FGF4) gene have previously been described in the domestic dog. An FGF4 retrocopy on chr18 is associated with disproportionate dwarfism, while an FGF4 retrocopy on chr12 is associated with both disproportionate dwarfism and intervertebral disc disease (IVDD). In this study, whole-genome sequencing data were queried to identify other FGF4 retrocopies that could be contributing to phenotypic diversity in canids. Additionally, dogs with surgically confirmed IVDD were assayed for novel FGF4 retrocopies. Five additional and distinct FGF4 retrocopies were identified in canids including a copy unique to red wolves (Canis rufus). The FGF4 retrocopies identified in domestic dogs were identical to domestic dog FGF4 haplotypes, which are distinct from modern wolf FGF4 haplotypes, indicating that these retrotransposition events likely occurred after domestication. The identification of multiple, full length FGF4 retrocopies with open reading frames in canids indicates that gene retrotransposition events occur much more frequently than previously thought and provide a mechanism for continued genetic and phenotypic diversity in canids

A RAPGEF6 variant constitutes a major risk factor for laryngeal paralysis in dogs.

Laryngeal paralysis (LP) is the inability to abduct the arytenoid cartilages during inspiration, resulting in a partial to complete airway obstruction and consequent respiratory distress. Different forms of LP with varying age of onset exist in dogs. Hereditary early onset forms were reported in several dog breeds. In most breeds, hereditary LP is associated with other neurologic pathologies. Using a genome-wide association study and haplotype analyses, we mapped a major genetic risk factor for an early onset LP in Miniature Bull Terriers to a ~1.3 Mb interval on chromosome 11. Whole genome sequencing of an affected Miniature Bull Terrier and comparison to 598 control genomes revealed a 36 bp insertion into exon 15 of the RAPGEF6 gene (c.1793_1794ins36). The imperfect genotype-phenotype correlation suggested a complex mode of inheritance with a major genetic risk factor involving a recessive risk allele. Homozygosity for the insertion was associated with a 10- to 17-fold increased risk for LP. The insertion allele was only found in Miniature Bull Terriers and Bull Terriers. It was absent from >1000 control dogs of other dog breeds. The insertion sequence contains a splice acceptor motif leading to aberrant splicing in transcripts originating from the mutant allele (r.1732_1780del). This leads to a frameshift and a premature stop codon, p.(Ile587ProfsTer5), removing 64% of the open reading frame. Our results suggest an important role of RAPGEF6 in laryngeal nerve function and provide new clues to its physiological significance

Phenotypic Effects of FGF4 Retrogenes on Intervertebral Disc Disease in Dogs.

Two retrogenes on chromosomes 12 (12-RG) and 18 (18-RG) contribute to short-limbed phenotypes in dogs. 12-RG has also been associated with intervertebral disc disease (IVDD). Both of these retrogenes were found to be widespread among dog breeds with allele frequencies ranging from 0.02 to 1; however, their additive contribution to disease is unknown. Surgical cases of IVDD ( = 569) were evaluated for age of onset, disc calcification, and genotypes for the retrogenes. Multivariable linear regression analysis identified the presence of one or two copies of 12-RG associated with significantly younger age at first surgery in a dominant manner. 18-RG had only a minor effect in dogs with one copy. Multivariable logistic regression showed that 12-RG had an additive effect on radiographic disc calcification, while 18-RG had no effect. Multivariable logistic regression using mixed breed cases and controls identified only 12-RG as highly associated with disc herniation in a dominant manner (Odds Ratio, OR, 18.42, 95% Confidence Interval (CI) 7.44 to 50.26; < 0.001). The relative risk for disc surgery associated with 12-RG varied from 5.5 to 15.1 within segregating breeds and mixed breeds. The FGF4 retrogene on CFA12 acts in a dominant manner to decrease the age of onset and increase the overall risk of disc disease in dogs. Other modifiers of risk may be present within certain breeds, including the FGF4 retrogene on CFA18